Path-121-Practical C

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PATH-121 - practical
Plant Pathology (Mahatma Phule Krishi Vidyapeeth)
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LABORATORY PRECAUTIONS
1. Each student should observe the cleanliness while working in the laboratory.
2. The laboratory instruments such as microscope, micrometers, electrically

operated instruments etc. should be handled carefully under the guidance of
teacher or laboratory assistant.
3. At the time of staining, care should be taken to avoid the spoilage of top of the
working table and floor.
4. Each student will be held responsible for the breakage or loss of any
equipment.
5. Student should attend the practicals with essential material viz., pencil, razor,
needle and piece of white cloth.
INSTRUCTIONS
1. All figures must be drawn with drawing pencil.
2. Get the practical record checked and signed regularly by the teacher in charge,
is the same or forthcoming practical.
3. Assessment of the practical will be done on the basis of attendance in the

practical and proper drawings.
4. If a student remains absent for a practical, the assessment of exercise will be

done as zero marks.

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INDEX
Ex. Page
Title of Exercise Date Sign
No. No.
1. Acquaintance with various laboratory
equipments and microscopy
2. General study of different structures of
fungi
3. Study of symptoms of various plant
diseases
4. Study of representative fungal genera
5. Staining and identification of plant
pathogenic bacteria
6. Study of phanerogamic plant parasites
7. Transmission of plant viruses
8. Study of morphological features and
identification of plant parasitic nematodes
9. Preparation of culture media.
10. Isolation and purification of fungi and
bacteria
11. Extraction of nematodes from soil
12. Koch’s postulates
13. Study of fungicides and their formulations.
14. Methods of fungicide application and their
safe use.
15. Calculation of fungicide spray
concentrations
16. Collection and preservation of disease
specimens

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EXERCISE NO.1
ACQUAITANCE WITH VARIOUS LABORATORY EQUIPMENTS AND

MICROSCOPY
Certain equipment or devices are required to perform even the fundamental
exercises in any discipline of biosciences. There are certain equipments which come in
the category of essentials and some equipment are those which are required for 12
specific tests. We would be discussing only the most commonly required devices, their
uses and precautions while using them.
1. HOT AIR OVEN:
It is a vertical steel box with double or triple walled body of aluminum or
stainless steel, partitioned with wire mesh trays inside. The body of the devise is provided
with heating element between the walls either at the bottom of the box (bottom heated) or
on all three side of the body (universal heating). The outside of the body painted with an
epoxy powder coating. Some ovens are also provided with system for circulating hot air
in between the inner chamber and insulation through forced air moved by motorized
blower so as to minimize the temperature variation at any point in working space. Ovens
are available in varying capacity. A thermostat control provided to maintain the
temperature inside with the sensitivity of ± 30C or less. The door is provided with
synthetic rubber gasket to make it air tight. The range of temperature inside varies from50
to 3000C or more. The front of the oven is provided with a digital temperature controller-
cum-indicator and power switch for on/off. In ovens, the temperature is maintained above
the ambient temperature. This devise is commonly used for dry heat sterilization of many
objects such as glass wares like Petri dishes, pipettes, flasks and other useable without
graduations, during many biological exercises. The temperature required for sterilization
may vary with the time of exposure to the dry heat, as mentioned below.
Temp. Time
1. 120°C 8hr
2. 140°C 3hr
3. 160°C 1hr
4. 180°C 20min
Precautions :
 The oven should not be too closely packed to allow the air to circulate. The heat
must circulate to all contaminated parts.
 It is best to wrap the material to be sterilized or keep it in containers to retain
sterility after treatment.

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 Time is counted once the desired temperature is achieved in the oven.

 Do not open the device immediately after the process is finished. It would cause
contamination of the material and the glass wares would break getting exposed to
cool air on immediate opening of the oven.
2. BOD INCUBATOR:
The biological oxygen demand (BOD) incubator maintains a range of temperature
below and above the ambient temperatures required for growth and multiplication of
various micro-organisms. It is a vertical steel chamber shaped as an atmirah made up of
double or triple walled body. Outer surface is painted. Incubators are available in varying
capacities. Temperature inside may be maintained from 5°C to 50°C with an accuracy of
±l°C. Incubator is provided with both heating and cooling systems. Heating may be of
two types. Bottom heated and universal heated (in which the heating element is placed in
all three side walls) with a thermostatic control while cooling is maintained by
compressors. It is provided with air circulation fans for uniform distribution of
temperature inside.
If required, fluorescence lights of 60cm may be installed, vertically along the back
wall of the device for illumination. These lights are incorporated with timer 0-24h for
regulating illumination period. An inlet nozzle may also be installed for monitoring
CO2/air mixture concentration inside and humidistat for control of humidity (55% to
95%) by natural mist outside, on the front surface it is provided with switches for
manual/automatic temperature controller, heat energy regulator, digital temperature
indicator, cooling/heating indicators and mains.
The cultures demand an ambient temperature, humidity and oxygen for its
isolation and multiplication. The required conditions are adjusted in the incubators
following the instructions for growth and multiplication of the organisms which may vary
from one organism to another. The culture plates are, thus, incubated for desired periods
in incubators.
Precautions :
 Always label the material while keeping in the incubator with mention of date of
placing and recording the observations.
 Frequent opening of the device causes variations in the maintained temperature.
 Incubators are cleaned and sterilized at frequent intervals to avoid contamination
of the materials.
3. LAMINAR AIR FLOW CABINET:

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The cabinet is fabricated out of thick board of sunmica or is of stainless steel.

Interior surface of working platform/table is of stainless steel with the sunmica clad at the
top. Sides of the panels are of thick transparent Plexi/acrylic glass duly framed. The unit
is fitted with both pre filter and high efficiency particular air (HEPA) filter. Air is drawn
through pre filter and is made to pass through highly effective HEPA filters having
efficiency rating as high as 99.99% thus retaining all the particles of size 0.3 micron or
larger. A blower and motor assembly of 1.5 HP, dynamically and statistically balanced is
fixed.
The working area is illuminated by fluorescent fight fitted with the unit. Height of
the working table is adjusted to be comfortable to sit down for operator. A UV light is
also fixed underneath the sun mica clad at the top and it is switched on 10-20 minutes
before working. Cock for gas, vacuum line is also provided at the outer layer of the top
clad,
Laminar flow provides an aseptic or micro-organism free environment for
performing various activities such as pouring of sterilized media in sterilized plates,
isolation and transfer of pathogens during application of different methodologies require
aseptic or sterilized environment
Precautions
• Proper care is taken not to expose any part of the body to the UV light as the exposure
may be carcinogenic or mutagenic.
• After every use, clean the platform.
• Keep the blower on, while working.
• Vacuum line is monitored time to time.
4. CENTRIFUGE:
Various methodologies require centrifugation of suspensions for separation of
various particles of different densities through centrifugal force. The body of the device is
equipped with microprocessor controller for regulating the speed in rpm. It has got an
autoclavable rotor with 12 positions for 15ml DIN tubes. The rpm varies in different
centrifuges. The simplest and commonly used is the table centrifuge which has 3000 rpm
and is generally used for washing test and for other routine work. Ultra centrifuge may
have a speed as high as 15,000 rpm or even more.
5. REFRIGERATOR/DEEP FREEZE:
There is need to maintain the cultures in pure form for further studies. The
maintenance of culture, in general, is carried out at low temperatures (0-5°C) because at
lower temperature all life processes slow down and culture may be maintained without
loosing their identity for a longer period. This is carried out in an refrigerator. Apparently,

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refrigerators resemble to the incubators but the basic difference is that the temperature
maintained inside refrigerator is always below the ambient temperature unlike incubators
where temperature may be maintained below and above the ambient temperatures
ranging from 5°C to 50°C.Deep freeze has all specifications similar to that of refrigerator
except that the temperature maintained is below 0°C. Therefore, puff insulation and more
than one compressor is installed.
6. AUTOCLAVE/STEAM STERILIZER:
Autoclave works on principle that the increase in pressure is directly proportional
to the increase in temperature. It is used for sterilization of various utilities specially the
media under saturated steam pressure at any selected point between 10-20 psi. Autoclaves
may be either vertical or horizontal cylindrical double or triple walled unit mounted on a
sturdy stand. The inner chamber (boiler) is made up of stainless steel and outer surface is
of mild steel duly painted. The heating element is fitted, at the base of the cylinder. The
space between the outer and steam jacket inside is insulated to minimize temperature
loss. The lid is fitted with rubber gasket and tightened by wing nut/ radial locking system.
All autoclaves are fitted with standard accessories such as water indicator,
pressure gauge, steam release cock, spring loaded safety valves.
The steam pressure is hydraulically maintained which helps to shoot up the
temperature quickly. Generally the steam sterilization is done at 15 p.s.i. for 15 minutes.
However, the time is counted after the pressure is achieved. Devices such as steam
sterilizers are also manufactured by some firms which help in intermittent sterilization for
inactivating the spores preferably in soil samples.
Moist heat sterilization is performed in autoclave under pressure. It has more penetrating
power than dry heat. Most media can be sterilized by heating at 10-15 p.s.i. (10-15 lb/in2)
for 15 min. in an autoclave or in domestic pressure cooker. Following are the
temperatures at various pressures:
p.s.i. Temperature (°C)
5 107
7 110
10 115
15 121
20 126
Precautions :
• Check that the sufficient water up to marked level is present in autoclave. Alt exhaust
vents and safety valves as well as the chamber should be kept clean.
• Screw down lid, tighten diagrammatically opposite wing nuts in pairs so that the lid is
clamped evenly on the gasket.

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• Open stream cock and light gas or switch on electricity.

• Let steam issue for at least 5 minutes to remove at) air before closing cock. This is
very important to obtain correct pressure and temperature.
• Sterilization time is calculated after the load has reached the desired temperature.
• Over sterilization should be avoided otherwise it may cause hydrolysis of the
compounds (carbohydrates etc.).
• Allow to operate for the required time and turn out gas or electricity.
• Let pressure drop to zero by cooling, than by opening steam cock.
• Wait 5 minutes and open lid.
• The use of non-absorbent cotton plugs which should be firmed enough to allow for
access of steam and air exhaust during decompression. "Acidification of the medium,
if required, is done after autoclaving.
• Vitamins, hormones, antibiotics and other such compounds may be destroyed by
heating and therefore should be sterilized by filtration or other means and added after
autoclaving the medium (remember that when liquids are mixed there is a dilution
factor which is to be considered).
7. WEIGHING BALANCE:
Many types of balances such as single pan balance, top loading electrical balance,
analytical balance, are available for weighing different ingredients required during course
of experimentation. The accuracy of weighing is determined by the sensitivity of the
balances which may be as low as 0.000lg, Electrical balances are easy to handle and are
more accurate and sensitive.
• Electronic balances are highly sensitive and even fans on may affect the accuracy of
the measurements.
• After every use clean the pan with dry clean tissue paper and keep in dust proof
chamber.
MICROSCOPY

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Microscope :
Principle : The lenses in this instrument are so adjusted that minute objects
invisible to naked eye are magnified and made visible.
There are two types of microscope in common use.
1. Simple microscope: Only one lenses (or one set of lenses) is used in
between the eye and object e.g. Hand lenses and dissecting microscope.
2. Compound microscope: Two or more than two lenses (or set of
lenses) are used in between the eye and object and this helps in obtaining more
magnification e.g. Student’s microscope, Research microscope.
Magnification: It is defined as enlargement in original size of the object brought about
by lens or lenses. The total magnification of an object is determined by the
multiplication of magnifying power of the eye piece by magnifying power of objective.
e.g. Eye piece (10x) objective (44x) = 440.
Mounting of cultures and specimens for microscopic investigation

Objectives:
1. To study the different methods of culture and specimen mounting under the
microscope for investigation.
2. To study the morphological characters of the microorganisms from the isolated
cultures in order to identify it for further studies.
3. To diagnose the cause of the disease.
1. MOUNTING OF CULTURES FOR MICROSCOPIC EXAMINATION
Most fungi are directly suitable for microscopic examination without previous
preparation or sectioning. An appropriate portion of a fungal preparation (culture) is
chosen using the naked eye or under a preparative, stereoscopic microscope and placed
into a drop of water on tile slide. So investigators use 0.1 M NaCl in distilled water. The
cover slip is "placed carefully" over the specimen and pressure applied according to need.
Under low magnification, the cover slip can be tapped to separate the fungus elements
Disturbing air bubbles in the preparation can be avoided if the following solution is used
instead of water:
Glycerol 10 ml
Distilled water 20 ml
90 per cent ethanol 30 ml
Arnann's solution

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Lactic acid 20 ml
Glycerol 40 g
Phenol 20g
Preparations made with water or Amann's solution can be preserved for months if
the edges of the cover slips are sealed hermetically. For this purpose a mixture of paraffin
and Vaseline (1:1) or other varnish, which dries quickly, can be used. Glycerol-gelatin
mounting medium is the most widely used for fixing fungus preparations on slides and
has the following composition
Gelatin 7g
Glycerol 50 ml
Phenol 1g
The boiled mixture is a dense, gelatinous substance but clear in its cooled state
and a cube is cut out for each mount and placed on a microscopic slide and melted above
a gas flame. The fungus part being investigated is put into the melted drop and it is
covered by a cover slip while still in a warm state. By pressing gently on the cover slip,
the surplus glycerol-gelatin medium is eliminated. Such fixed preparations obtained in
this way are preserved for a long time

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Instruments used in Laboratory of Plant Pathology and their uses
Sr. Name of Instrument Use

No.
1 Inoculating Needle Used in isolation procedure to transfer the fungal/ bacterial
colony from one Petri plate to another.
2 Petri Plate Used for isolation, multiplication and maintenance of fungal
or bacterial cultures. It was named after German bacteriologist
Julius Richard Petri, who invented it when working as an
assistant to Robert Koch
3 Test Tube Used for preparing slants of media. Also used in serial dilution
method for isolation of fungi or bacteria
4 A Bunsen burner It produces a single open gas flame, which is used for heating,
sterilization, and combustion.
5 Microscope To observe and examine the microorganisms
6 Agar Agar powder Used as solidification agent in different media
7 Dextrose Used as a source of sugar in different media
8 Conical Flask Used in preparation of media.
9 Sprit Lamp To sterilize the needle, cork borer etc
10 Cork Borer To prepare the holes in fungal colony in Petri Plate
11 Cotton To plug the conical flask and test tubes
12 Glass Slide To mount the culture for examination under microscope
13 Cover slip To cover the culture on glass slide
14 Forceps To transfer the bits of samples on media during isolation
procedure
15 Spreader To spread the cultures of pathogen on media.
16 Digital Colony Used to count the number of colonies of bacteria on a
Counter medium.
17 Micro pipette To take the exact quantity of solution in different procedures
like isolation, serial dilution etc.
18 Water Distillation To obtain the distilled water for different studies.
Unit

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EXERCISE NO. 2
GENERAL STUDY OF DIFFERENT STRUCTURES OF FUNGI
I. STUDY OF TYPICAL FUNGUS :

Fungi are Eukaryotic, achlorophyllus, uni or multicellular have a definate cellwall
makeup of either chitin or cellulose, intake food by means of absorption, reproduce by
means of production of sexual and asexual spores. The asexual reproduction proceeds by
means of production of vegetative spores. The sexual reproduction is by means of
production of male and female gametangia unite to form a zygote. The body of fungus
generally referred to as thallus. They are classified under Kingdom – Fungi (Ainswarth,
1973). Recently they are also classified under Kingdoms - Fungi, Stramenopila and
Protists (Alexopolulos et al., 2004).
Constituents Required:
Microscope, Slides, Cover slips, two pointed needles and wash bottles.
Procedure :
Take a piece of bread, moisten it and keep in moist chamber. After 48 hours
threadlike whitish growth appears on the bread. Take a thread like growth on a clean
glass slide with the help of pointed needles; spread the threads in the drop of water on
slide. Place the cover slip gently over the water drop so that no air bubbles are let inside.
Observe first under low power and then focus under high power of the microscope.
Observations :
Look for an individual thread known as Hypha. Many of such hypha constitutes
a Mycelium. Observe, if cross walls are present in the hypha. The cross wall is called a
septum or septa. The mycelium having septa is called septate mycelium. A mycelium
without Septa is called aseptate, non septate or coenocytic mycelium. Observe also
for sporangiophore, sporangia, sproes, rhizoides, sporangiole, stolon, columella etc.
Sporangiophores :
A specialized hyphae upright in growth produced from the mycelium, bearing sac
like spore fruit or structure is known as sporangiophore.
Sporangium : Sporangium is a sac like structure or spore fruit containing spores.
Sporangiospores or spores : Sporangiospores or spores are the unit of reproduction,
round to oval, hyaline, unicellular and produced internally or endogenously.
Columella : The knob like structure at the end of sporangiophore over which the
sporangium is attached. It is an attachment between sporangium and sphorangiophore.
Rhizoids : The root like structures or appendeges of fungus are known as rhizoids. It
has two main functions is anchoring or to hold fast with main host and absorption of food
material.

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Sporangiole : A small sporangium without columella.

Stolon : The hypha which joins two rhizoids is known as stolon.
Also observe a culture of fungus provided and note the type of mycelium,
conidiophores and conidia.
Septate mycelium : Hypha divided by cross walls (Septa)
Conidium : Asexual spore borne on the hypha (Conditiophore) is called ‘conidium’.
Conidiophores : The hypha bearing the conidium.
II. VEGETATIVE STRUCTURES :

Morphology of fungi includes both vegetative and reproduction structure of fungi.
A fungus body generally consists of thread or filaments. An individual thread is called
hypha. A group of hypha is known as Mycelium. Examine the suitable preparation and
record the following types of mycelium within fungi.
Non-septate or aseptate or coenocytic mycelium
The mycelium whose hypha have no cross walls or septa and which consists of
number of nuclei embeded in cytoplasm. So the mycelium is multinucleate or uninucleate
e.g. Fungi belonging to sub division Zygomycotina and Mastigomycotina.
Septate mycelium
The hyphal cells of the mycelium are divided into the compartments by cross
walls or septa such mycelium is called septate mycelium. Individual cell may contain
one or more nuclei depending upon species hence mycelium may be uni or multinucleate
e.g. Fungi belogning to sub division Ascomycotina, Basidiomycotina and
Deuteromycotina.
Ectophytic mycelium
The hypha in this case grow on the external surface or on epidermal cells,
produces special sucking organs or structures called as haustoria e.g. powdery mildew of
pea, grape and cucurbits etc.
Endophytic mycelium
When the hyphae of a mycelium grow inside the epidermal layer of plant tissues,
it is called endophytic mycelium. Endophytic mycelium is of following types.
i) Intercellular ii) Intracellular iii) Vascular.
Intercellular mycelium : When the mycelium is found to grow in between two cells of
plant tissue without penetrating the cells and obtain nourishment by sending or haustoria
in the cells, it is known intercellular mycelium e.g. Stem, Leaf, Yellow rusts of wheat,
Jowar rust, Bajra rust etc.
Intracellular mycelium : When mycelium grows within the cells of the plant tissue, it is
called intracellular mycelium i.e. smut diseases viz. Different Smuts of Jowar, Bajra,
Sugarcane smut etc.

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Vascular mycelium : When the mycelium is confined to or found to grow in vascular

tissues of plant, it is called vascular mycelium e.g. Fungus causing wilts viz. Pigenopea
wilt, Cotton wilt, Chickpea wilt, Safflower wilt etc.
III. MODIFICATIONS OF MYCELIUM

Sclerotium : (Pl. sclerotia) (Skeron hard). Sclerotia are the modified forms of vegetative
mycelium, which form hardened compact mass of the hyphae and act as resting body
which is resistant to unfavourable conditions and may remain dormant for long periods
and germinate under favourable conditions e.g. Sclerotium spp.
Rhizomorphs : (Rhizo-root + Morphe – Shape)
A number of fungi produce thick cable like strands made up of hyphae where the
hyphae have lost their individuality. These grow either in soil or on the trunks of the
trees. They can withstand unfavourable conditions. They are found in fungi belonging to
subdivision Basidiomycotina.
Stroma : (Pl. stromata)
It is a compact mass of the hyphae and appears as pseudoparenchymatus tissue
and contain fruiting body of the fungus e.g. Ergot sclerotia.
Chlamydospores : These are the thickened or swollen cells of the mycelium containing
stored food material and may be formed terminally or intercalary. These are the resting
cells which withstand the unfavourable conditions and germinate during favourable
conditions e.g. Fusarium sp.
Dormant mycelium : It is the mycelium which hibernates in the host tissue to tide over
unfavourable conditions. It remains in a dormant condition for a part of its life cycle and
became active when conditions are favourable e.g. Downy mildew of grape, Kolegroga
of arecanut and loose smut of wheat.
Gemmae : (Pl. Gemma) :
These are the chlamydospores produced in lower fungi whose walls are thinner
e.g. Saprolegnia sp.
EXCERCISE:
1. Draw a neat diagram of a typical fungus and lable all the parts ?
2. What is a fungus ? Give its position?
3. What is the initial source of mold on the bread ?
4. What are the conditions required for proper growth of bread mold fungus
5. Draw neat diagrams of different fungal vegetative structures.
6. What is the role of haustoria, Sclerotia and chlamydospores in the life cycle of fungi?

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REPRODUCTIVE STRUCTURES AND SPORE FRUITS IN FUNGI

Objectives:
1. To acquaint with types of reproduction in fungi.
2. To know about different fruiting structures produced by fungi which help
repetitive life cycles and hibernation of the pathogens.
The fungi reproduce by spores. Spore is a minute reproductive or propagative unit
functioning as a seed of fungi. These are produced in three ways.
i) Asexually ii) Sexually iii) Vegetatively.
SPORES
Asexual Sexual Vegetative
Endogenous Exogenous Zygote Chlamydospores

e.g. Conidia Zygospores Arthrospores
Oidia Oospores
Ascospores
Basidiospores
Motile Non Motile
Zoospores Aplanospores
ASEXUAL SPORES IN FUNGI
Asexual spores develop or form without nuclear fusion or act of breeding and
these spores mainly borne on sporophores. They are not usually resistant to unfavourable
conditions. They are capable of rapid multiplication, and are well adopted for efficient
dissemination. They may be one or many celled, borne on the specialized hyphae or
produced in special structures called as spore fruits.

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Endogenous:
These spores are formed internally within enlarged cell or sac (sporangium) by
division of protoplasm. e.g. sporangiospores.
Sporangiospores :
The sporangiospores are produced in a enlarged cell or sac or sporangium and are
unicellular. These spores liberated by breaking the wall of sporangium. When
sporangium gives motile spores it is known as zoosporangium or swarmsporangium
and the spores as zoospores, or swarmspores. These spores are motile by means of the
flagella or cilia. A non motile spore produced in the sporangium is known as
aplanospore.
Exogenous:
These spores are borne externally on sporophores.
e.g. conidia, oidia, etc.
Conidia (Sing. Conidium) :
The conidia are produced asexually on a specialized hyphae or stalk known as

conidiophore and spores are called as conidia. Condia differ in their size, shape, colour,
septation and branching within the same species. Conidia may be uni, or multicellular,
e.g. Alternaria, Pyricularia, etc.
Oidia (Sing. Oidium)
These spores are barrel shaped or rectangular in shape and are produced asexually
in chains on the stalk called as oidiophores, e.g. Oidium mangiferae - oidia in powdery
mildew of mango.

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1. Coremium or Synnemata (Pl. Coremia):
The hyphae, which form conidiophores and erect conidiophores, grouped together to
form coremia. Each coremium consists of sterile stalk terminating into fertile hyphae
bearing conidia, e.g. Stysanus thyrosoides.
2. Sporodochium (Pl. Sporodochia):
A spore fruit having cushion shaped stroma covered with the conidia formed inside ooze
in sticky mass is known as sporodochium, e.g. genus Nectria (Sporophyte fungus
growing on the trunks).
3. Pycnidium (Pl. Pycnidia):
Spherical or oval shaped spore fruit with short conidiophores lining inner side, which
bear spores or conidia called pycnidiospores. The spore fruit usually have an opening is
called ostiole, e.g. Phoma spp., Phomopsis spp. etc.
4. Acervulus (Pl. acervuli):

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It is compact mass of hyphae giving rise to short, simple, hyaline
conidiophores, closely packed together forming cushion like mass with or without setae.
It is also known as modified open sorus, e.g. genus, Colletotrichum and Pestalotiopsis.
5. Sorus (Pl. sori):

It is a little heap like compact mass of sporophores and
spores, which usually are covered by epidermis. At
maturity, the epidermis breaks and all the spores are
liberated, e.g. smut and rust.
6. Aecium (Pl. aecia) :

It is an inverted cup like or bell shaped structure usually
formed on lower surface of the leaf, consisting of binucleate
hyphal cells producing yellow or orange coloured spores, which
are usually formed in basipetal manner called aeciospores, e.g. rust
fungi, aecium of Puccinia graminis tritici.
7. Pycnium or Spermagonium (Pl. Pycnia) :

It is characteristics of rust fungi. A flask shaped structure
containing pycniosproes or spermatia, e.g. rust fungi.

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SEXUAL SPORES IN FUNGI
The sexual spores are formed by the fusion between two gametes of opposite sex.
These spores are generally produced under adverse conditions. Cell carrying the gamete
is called gametangium and gamete is unisexual or haploid.
Depending upon the manner of formation of spores they are classified as zygote,
zygospore, Oospore, Ascospore and Basidiospore and can resist the unfavourable
conditions.
Zygote : Zygote is formed by the union of two opposite haploid motile gametes, e.g.
lower fungi of the phylum Chytridiomycota, class Chytridiomycetes.
Oospores:
It is the result of union between female and male

gametes i.e. oogonium and antheridium,
respectively. Oogonium is larger than antheridium
and is oval or irregular. Anthridial nuclei passes to
oogonium through fertilization tube. The oospores
are thick walled and may be smooth or rough, dark
brown in colour. These spores can resist the adverse conditions, e.g. phylum Oomycota,
class-Oomycetes, order Peronosporales (e.g. Downy mildew of grape, etc.)
Zygospores:
Zygospores are formed by the union of two

similar or identical gametes designated as + ve
and –ve. The resultant spore is thick walled
echinulated or spiny. The wall consists of two
layers. Outer one is known as exosporium and
inner layer as endosporium. These sproes resist
unfavourable conditions and germinate during favourable season. e.g. fungi of the

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phylum Zygomycota, class-Zygomycetes and Trichomycetes, order Mucorales and

Entomophthorales.
Ascospores :
Ascospores are produced in a sac known as ascus which

is formed in fruiting body, e.g. Perithecium and
Cleistothecium. Ascospores in the ascus are generally
eight in number but it may vary and always in multiple
of ‘2’. The ascospores are produced by union of male
and female gamete, i.e. antheridium and ascognoium
through fertilization tube (Trycogene). Ascospores may be single or many celled, hyaline
or coloured and having various shapes, e.g. fungi belonging to phylum Ascomycota.
Basidiospores:
These are the sexual spores produced on club shaped

structure known as basidia (basidium) on a short tube
known as sterigmata. Usually four basidiospores are
produced on each basidium. The basidiospores are
exogenously formed spores. In these fungi sexual
organs are absent, except in rust fungi, e.g. phylum –
Basidiomycotina.
SEXUAL SPORE FRUITS
ASCOCARPS:
It is the spore fruit produced by the fungi belonging to the phylum Ascomycota. Sexual
spore produced endogenously are known as ascospores in sac like structure called ascus (Pl -
Asci). The spore fruits are of various forms and shaped viz., spherical, flask, cup, saucer, pod, etc.
Following are the different types of ascocarps :
1. Cleistothecium (Pl. cleistothecium):

It is closed without ostiole, round to oval ascocarp with
irregularly arranged or scattered asci having dark brown to
black colour and provided with appendages to anchor or to
hold host and to help in dissemination. Cleistothecium
breaks open at maturity by wear and tear, e.g. powdery
mildew fungi of order Erysiphales.

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2. Perithecium (Pl. Perithecia):
A flask shaped
ascocarp with narrow neck like
ostiole through which asci are
released. The sterile structures
present in between the asci
within the ascocarp are known as
paraphyses, which help asci
in nutrition and dispersal, e.g.
Claviceps, Glomerella, etc.
3. Apothecium (Pl. Apothecia):

A cup or saucer shaped spore fruit with broad opening
is known as apothecium. The asci are arranged in
palisade layer called hymenium. The apothecium is
usually fleshy and leathery in nature, e.g. genus
Sclerotinia.
4. Ascostroma:
The asci are formed directly in a locule or cavity within at stroma. The stroma
forms the wall of the ascocarp.
BASIDIOCARPS:
These are the fructifications of phylum Basidiomycota and consist of mushroom,

bracket fungi and puff balls. They are highly developed and have a compound structures,
may be fleshy, leathery, woody or waxy in nature and bear special structures variously
known as gills, pores, needles and chambers. The sexual spores are the basidiospores,
produced exogenously on the tip of enlarged cell of hypha known as basidium (basidia).
The basidiospores are usually 4 in number. The basidia are intermingled with sterile
strucutres called paraphyses. The cells slightly larger than basidia intermingled in
hymenial layer are known as cystidia.
1. Puff balls :

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It is round or spherical, very small to big basidiocarp,

commonly found on dead organic matter. The
basidiospores are produced in the hymenium which lines
the inner surface. On maturity basidiospores are given
off, in the form of puff or smoke. The puff balls
have got small stalk at the base.
2. Bracket fungi :
A compound fruictification growing on dead tree
trunks. These are woody and hard basidiocarps. They
are typically bracket, hoof or saddle shaped, and highly
coloured with short stalk. The hymenial layer is found
on the honey comb fashioned pores in which basida
and basidiospores are observed.
3. Mushrooms :
These are the fleshy or leathery compound
fructifications with variously coloured, commonly found on
manure pits, dung heaps and on any rich organic matter.
They are borne on stalk and provided with gills and pores
to the underside which contains hymenial layer. The
mushroom may be edible and non-edible or poisonous, e.g.
Agaricus sp. (edible).
VEGETATIVE SPROES
Chlamydospores:
These sproes are
formed from hyphal
cells of old
mycelium enveloped
by a thick cell wall,
which later on
separate from parent hyphae and behave as resting spores. They may be formed
terminally or intercalary, e.g. Fusarium, Phytophthora, etc.
QUESTIONS
1. Draw neat diagrams of different fungal spore fruits.
1. Enlist asexual and sexual spore fruits.
2. What is the difference between sexual and asexual spores?
3. What is a gamete ?
4. What is the function of chlamydospores?
5. What are the different methods of sexual reproduction in fungi?

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EXERCISE NO. 3
STUDY OF SYMPTOMS OF VARIOUS PLANT DISEASES
SYMPTOMS: Symptoms are expressions of diseased conditions. They are expressed

internally as well as externally and help in general diagnosis. With the help of symptoms
a diseased plant can be identified from a healthy one. However, symptoms alone are not
helpful in ascertaining the exact nature of the disease. Similarly, symptoms may result
from different causes, unrelated to each other e.g. Chlorosis may be due to downy
mildew, viral infection or deficiency like the fever in human being may be due to wound,
typhoid or by malaria.
SIGNS : Signs are the experimental or scientific evidences of the diseases and generally
confirmed by various diagnostic techniques. Signs help in accurate diagnosis of the
diseases. Signs are the actual presence of the pathogen or its structures on the host or in
the host as a result of manifestation. e.g. presence of whitish growth on the leaves in
downy mildew of grape or jowar, bacterial ooze in ring disease of potato etc.
SYMPTOMS OF PLANT DISEASES PRODUCED BY FUNGAL PLANT
PATHOGENS:
1. MOTTLING : Partial destruction of chlorophyll in interveinal area e.g. Mottle
leaf of citrus.
2. STEM GALLS : e.g. white rust of crucifers, Loranthus on mango.
3. CLUB ROOT : e.g. club root of cabbage
4. BLIGHT : There is a general and rapid destruction of plant parts like shoots,
leaves, blossoms, twigs etc. the dead organ turn as brown to black showing burnt
appearance e.g. Early and late blight of potato, Bacterial blight of paddy.
5. SPOT : It is localised destruction of the tissue in a more or less circular manner.
It is usually found on the leaves, and may develop on stem or fruit. The dead tissues
which are in limited area give shapes as angular, round or circular surrounded by
yellow purple red margin e.g. eyespot of jowar, tikka of groundnut, angular leaf spot
of cotton.
6. TAR SPOTS AND STREAKS OR STRIPES : Necrotic area become typically
tar stained found in forest trees, palms, grasses and jowar. Streaks are elongation of
necrosis e.g. bacteiral streak of paddy and jowar.

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7. BLAST : Same as blight but spots are distinct and spindle shaped e.g. blast of
paddy.
8. DIE BACK : Dying of plant organ especially stem and branches from the tip
downward e.g. die back of citrus.
9. EXUDATION : Secretion of sticky gum like substance due to diseases e.g.
gummosis of citrus.
10. ANTHRACNOSE : Distruction of collenchyma and cambium tissue, lesions are
sunken in the centre with raised and prominent margin e.g. anthracnose of grape,
chilli and bean etc.
11. BLACK HEART : Blackening of central portion observed in potato due to high
temperature and poor ventilation in storage e.g. black heart of potato.
12. SCAB : Destruction of epidermal tissues in the form of scab. Infection is deep
seated e.g. scab of potato and apple.
13. SHOT HOLE : Decayed leaf tissues are blown away leaving holes or
perforations e.g. shot hole of ashok and mango.
14. SMUTS : The floral parts are usually , affected the ovaries destroyed and
replaced by forming sori e.g. smuts of jowar, loose smut of wheat etc.
15. RUSTS : The pustules of spores usually breaking through the epidermis are seen
on the host. Pustules may be either dusty or compact and white, yellow, brown, red
or black in colour e.g. white rust of crucifers, leaf rust and stem rust of wheat
16. ERGOT : Normal grains are replaced by sclerotia e.g. ergot of bajra.
17. GREEN EAR (Downy Mildew) Flowers are converted into green and elongated
diseased structures e.g. green ear of bajra.
18. POWDERY MILDEW : Powdery growth consisting of mycelium and
numberous conidia is seen on the host surface e.g. powdery mildew of pea.
19. MUMMIFICATION : These are observed in fruits. The skin of fruit becomes
hard and fruit gets shrivelled such fruits are called as mummified fruits e.g. downy
mildew of grape.
20. WILTS : Wilting or drying of entire plant observed in adult plants. The leafes
and other succulent parts loose turgidity become flaccid and droop. It is typical
vascular symptom due to plugging of xylem vessel or toxic effect e.g. tur wilt, cotton
wilt, pea wilt, gram wilt etc.

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21. DAMPING OFF : Sudden wilting and collapse of seedlings observed commonly
in seed beds. The stem near the soil is affected, becoming constricted and weak e.g.
damping off of seedlings like tobacco, tomato, cabbage, chili etc.
22. Pallor : Partial destruction of chlorophyll in the form of streaks. There is un
healthy appearance of the plant due to deficiency or excess of water or lack of light
or reduction in chlorophyll content due to pathogenic organisms. e.g. bajra
seedlings affected with downy mildew.
23. ROTS : The term is applied in cases where affected tissue decays or rots.
Infection of parenchyma, pitch tissues and various parts. Rot imparts different
colour reactions and are designated accordingly.
a) Dry rot : Decay of tissues, even after rotting may sometimes remain firm or hard e.g.
dry rot of potato and corn.
b) Soft rot : Decay of soft tissue, rotting accompanied by softening of the tissue, e.g. soft
rot of lemon, mango, tomato, banana etc.,
c) Red rot : Affected tissues become red in colour e.g. red rot of sugarcane.
d) Wet rot : In addition to softening, there is slimy oozing of liquid e.g. storage rot in
potato, citrus and other fruits, usually due to fungi.
e) Root rot : Destruction of parenchyma of underground stems e.g. Rhizoctonia root rot
of cotton, hallow stem of jowar.
Rots may be described sometimes according to plant part affected e.g. stem rot
(Papaya), collar rot ( Groundnut) neck rot (Paddy), rhizome rot (ginger). Also they are
described after the discolouration produced on infection e.g. brown rot (Potato) black rot
(cabbage), red rot (Sugarcane) etc.

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SYMPTOMS OF PLANT DISEASES PRODUCED BY BACTERIAL PLANT

PATHOGENS:
1. TUMORS AND GALLS : Tumors are knot like structures or over growth of the
host tissue. It is bigger in size e.g. tumor caused by the infestation of bacteria like
Agrobacterium radiobacter. Galls are abnormal swelling or blisters or pimples /
knot formed on plant parts. The bacteria induces formation of galls in plants by
stimulating mature cells to resume meristematic growth, gall are smaller in size
than tumors.
2. HAIRY ROOT : Formation of numerous fine roots e.g. infestation of
Agrobacterium radiobacter var. rhizogenes.
3. WILTS : Wilting or drying of entire plant observed in adult plants. The leaves
and other succulent parts loose turgidity become flaccid and droop. It is typical
vascular symptom due to plugging of xylem vessel or toxic effect e.g. bacterial
wilt of tomato
4. BLIGHT : Here there is a general and rapid destruction of plant parts like
shoots, leaves, blossoms, twigs etc. the dead organ turn as brown to black
showing burnt appearance e.g. bacterial blight of paddy.
5. SOFT ROT : The term is applied in cases where affected tissue decays or rots.
Infection of parenchyma, pitch tissues and various parts. Rot imparts different
colour reactions and are designated accordingly. e.g. brown rot (Potato) /Soft
black rot (cabbage), etc.
6. CANKERS : Deep seated infection due to destruction of woody tissues and
cambium tissues. Cankers are raised from epidermal surface of the tissue and are
rough to touch e.g. citrus canker, guava canker etc.

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SYMPTOMS OF PLANT DISEASES PRODUCED BY VIRAL AND

PHYTOPLASMAL PLANT PATHOGENS:
I. COLOUR CHANGE IN LEAVES
1. Chlorosis: It is also known as yellowing. there is complete destruction of
chlorophyll. when the colour becomes white it is known as etiolation. These
symptoms usually caused by viruses, e.g. yellowing of beans.
2. Vein clearning/banding: Clearing of veins i.e they turn yellow and leaf lamina
remaining green, e.g. yellow vein mosaic of bhendi and hibiscus.
3. Flecks: Clearing of veins further turn into translucent appearance, e.g. tristeza virus
in kagzi lime
4. Mosaic: Mosaic caused by virus infection are highly infectious. it is due to partial
loss of chlorophyll or chlorosis in uneven patches, e.g. papaya mosaic, tomato
mosaic, chilli mosaic, etc.
a) Yellow mosaic: Light green and yellow patches are observed in the leaf
lamina, e.g. yellow mosaic of beans.
b) Streak: induction of the streaking on the infected portion mainly on the leaves,
e.g. maize streak.
c) Mottling : Partial destruction of chlorophyll in interveinal area, e.g. mottle leaf
of citrus.
5. Ring spots: The formation of the characteristic chlorotic or necrotic rings on the
leaves sometimes on fruit and stem, e.g papaya ring spot disease.
6. Oak leaf pattern: Yellow concentric lines extending along main veins, e.g. potato
aucuba mosaic virus.
7. Browning of leaf from tip downward: e.g. rice tungro virus
II) ABNORMALITIES OF LEAF SHAPE

1. Enations and tumours: Dark green tumor like outgrowth appears on the upper or
lower surface of leaves (enation), e.g. pea enation mosaic.
2. Leaf curl: Leaves curl upward or downward, e.g. leaf curl of chilli,
tobacco,papaya, brinjal, tomato, etc.
3. Leaf roll: Leaves roll upward or downward, plants remin stunted and have stiff
upright growth, e.g. potato leaf roll, etc.
4. Fern leaf and shoe string effect: Leaf famina between veins is poorly developed
or not developed at all, e.g. CMV on tomato, etc.
5. Cupping of leaves: e.g. papaya mosaic, cow pea mosaic, etc.
6. Twisting and blistering of leaves: Uneven growth of leaf lamina, e.g. TMV,
CMV in tomato, etc.

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III) ABNORMALITIES IN LEAF SIZE: Reduction in leaf size, e.g. CMV on tomato
IV) NECROSIS: Scattered flecks or patches of dead tissues appear on infected tisuues
of leaves, stem, fruits, etc., e.g. tomato spotted wilt virus, potato virus X and Y,
etc.
V) ABNORMALITIES IN STRUCTURE AND SHAPE OF PLANTS
1. Stunting/dwarfing (Bushy appearance): Reduction in size of leaves, flowers,
fruits, shortening of internodes and height which results into stunded growth of
plant, e.g. bunchy top of banana, pea stunt, etc.
2. Hairy root and spindle tuber: The formation of spindle tuber of potato due to
infestation of potato spindle tuber virus.
3. Swollen shoot: Virus inducing the swollen shoot and the branches, e.g. cocco
swollen shoot.
VI) SYMPTOMS ON BARK AND STEM:
1. Bark scaling: e.g. Citrus psorosis.
2. Cracking of bark: e.g. Citrus exocortis.
3. Stem pitting: Pitting and groving of the stem, e.g. citrus tristeza.
VII) SYMPTOMS ON FLOWERS:
Colour breaking (petal or flower break): Colour break symptoms which induces
varigation in the colour of flower, e.g. tulip flower mosaic, pea mosaic.
VIII) SYMPTOMS ON FRUITS:
1. Mottling of fruits: e.g. CMV in cucumber,
2. Watersoaked rings: e.g. Papaya mosaic,
3. Sunblotch of fruits: e.g. citrus greening in mosambi.
SYMPTOMS OF PHYTOPLASMAL PLANT PATHOGENS
1. Phyllody : The symptoms marked by vein clearing, stimulation of the axillary
buds and transformation of the flower parts into leafy structures termed as
phyllody, e.g. sesame phyllody.
2. Grassy shoot : Excessive tillering at the base of infected plants and grassy
transformation of the growth, e.g. grassy shoot in sugarcane.
3. Greening : Marked by yellowing of the midrib and lateral veins of mature leaves,
vein banding, distortion of leaves and blotching on the fruits, e.g. citrus greening.
4. Little leaf: Extreme reduction in the size of the leaves and leaves become sessile,
thin, soft glabrous and pale green, e.g. little leaf of brinjal.
5. Sandal spike: The symptoms are marked by severe reduction of leaf size and
shorting of the internodes as a result leaves become stiff and crowded giving
spiked appearance, e.g. sandal spike.
6. Stunting and dwarfing (Bushy appearance): Reduction in the plant size, leaf
lamina, node and internodes because of the infection of the phytoplasmal plant
pathogen, e.g. rice and barley yellow dwarf. In case of rice yellows disease
induced by the phytoplasma show profuse tillerning and pronounced stunting
occurs.
*******

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EXERCISE NO. 4
STUDY OF REPRESENTIVE FUNGAL GENERA

Objective: To study the morphological characters of the genera for their characterization
and disease diagnosis.
I. Pythium (Edson) Fitzpatrick

This is an aquatic organism responsible for causing the extremely destructive
disease like damping off. The pathogen can be isolated on the baits in the water.
Morphological characters: Mycelium hyaline, coenocytic; sporangia globose to oval,

terminal or intercalary on the somatic hyphae; oogonia globose, terminal or intercalary;
antheridia small, elongated or club shaped; oospores thick walled.
Material
 Affected specimen or culture of the pathogen, Microscope, Glass slides,

Cover-slips, Cotton blue,Lacto phenol, Razor blade, Hair brush, Inoculating
needles.
Procedure
1. Note pale green colour of the toppled-over seedlings. Examine the basal portion
of the stem and notice brownish, water-soaked lesion and the rotting tissues. Cut
sections of the affected areas, stain and examine under the microscope. Note the
killed host cells and collapsed tissues. Locate inter-and intracellular hyphae,
sporangia, oogonia, antheridia and oospores.
2. Take the culture of the pathogen, gently lift the small portion of the growth of the
pathogen with the help of the needle, mount on the glass slide and examine under
the microscope.
II. Phytophthora (Mont.) de Bary
The genus is responsible for causing the late blight disease of potato and was the
cause of epidemic in Ireland during 1845. It continues to be a major disease in the cool
and humid regions. In India, the disease is prevalent in the Nilgiri Hills, Bihar, Assam,
Bengal and plains of North India. In Maharashtra, it causes serve damage to potato crop
in Mahabaleshwar and Pachgani area.
Morphological characters: Mycelium coenocytic, sporangiophores branched with side

branches showing bulbous enlargements; sporangia hyaline, lemon-shaped with a papilla
at apex. Sporangia germinate by a germ tube or by zoospores but never form vesicle.

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Procedure
1. Cut a small portion of the infected leaf. Boil in 3-4 ml. of KOH solution. Place on
the slide with lower epidermis facing up and examine under low power. Watch the
sporangiophores emerging out of the stomata. Look for the attached sporangia.
Scrape an infected leaf. Mount the scrapped material in Lacto-phenol and study
the characteristic branching of sporangiophores and the typical shape of
sporangia. Brush out sporangia from the infected leaf, place in a drop of water on
the glass slides. Keep the slides in 2 moist chambers. Place one moist chamber
each in, a refrigerator at 100C and in an incubator at 24 0C. Observe the type of
germination at both the temperatures after intervals of 3 hrs.
the microscope
III. Albugo candida (Lev.) Kunze
The genus attacks the many species of Cruciferous. In cabbage, cauliflower and
radish seed crops, it induces the hypertrophy and hyperplasia of branches and floral parts
that cause heavy losses.
Morphological characters: Mycelium coenocytic; haustoria knob-shaped;
sporangiophores club-shaped; sporangia hyaline, globose, in chain; oogonium globose,
terminal or intercalary; antheridium clavate, paragynous; Oospores thick-walled.
Procedure
1. Note the symptoms and scrape the pustules with powdery consistency, mount in
Lacto-phenol and examine sporangia under low-power.
2. Cut cross sections of the leaves and hypertrophied organs, stain and observe under
the microscope. Observe intercellular mycelium, intracellular haustoria, short
sporangiophores arising from the mycelium in a compact layer beneath the
epidermis, chains of sporangia and the sexual organs and oospores.
the microscope
IV. Sclerospora graminicola (Sacc.) Schroet
This is one of the most important pathogen responsible for causing downy mildew
of pearl millet. There are two distinct stages of the disease. One is the downy mildew
stage and the other is the green ear stage.
Morphological characters: Mycelium coenocytic, intercellular; haustoria small,
bulbous; sporangiophores hyaline, broad, nonseptate, unbranched in the lower part with a
few short, thick branches formed dichotomously at the end; sporangia hyaline,
thin-walled, broadly elliptic with a papilla at end; oospores brown, thick-walled, covered
by an irregular, brown oogonial wall.

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Procedure
1. Examine chlorotic streaks on the upper surface of infected leaves and the downy
growth of the pathogen on the lower surface. Cut sections of the leaf, stain and
examine under the microscope. Note intercellular mycelium, intracellular
haustoria, sporangiophores emerging through the stomata and sporangia at the tips
of short branches of sporangiophores. Tease the leafy structure and examine the
oospores.
V. Plasmopara viticola (Berk. & Curt.) Berl. & De T.
Plasmopara viticola is responsible for causing downy mildew of grapes, causes huge
losses to the wine industry in France between 1878 and 1885. The disease still remains a
very important one in many grape-growing regions.
Morphological characters: Mycelium coenocytic, intercellular; haustoria globose,

intracellular; sporangiophores monophonically branched along the main trunk and
dichotomously branched at the right angle; sporangia hyaline, ovate; oogonia, spherical
formed terminally; antheridia clavate; oospores with thick smooth endospore wall
overlaid by a thinner rough exospores wall covered with the collapsed oogonial wall.
Procedure
1. Scrape the downy growth from the lower surface of a leaf, mount and examine
under the microscope. Notice the characteristic branching of sporangiophores and
hyaline sporangia.
2. Cut very thin section of the infected leaves, stain and examine intercellular
mycelium, intracellular haustoria and the sexual reproductive structures of the
pathogen.
VI. Mucor remosissimus
This is the most common occurring on the dead and decaying matter. It is
common on the bread hence also called as the bread mould.
Morphological characters: Mycelium coenocytic with stolon and rhizoid absent;
sporangiophore bearing a terminal sporangium; columella spherical; ripe sporangiospores
mostly ovoid; gametangia similar in size and shape; zygospores covered with a black,
thick, several-layered wall.
Procedure
1. Mount the whiskery growth from the bread and examine under the microscope.
Note mycelium, rhizoids, stolons, sporangiophores, columella, sporangia,
sporangiospores, gametangia and zygospores.
2. Take the small amount of the growth from the pathogen culture mount it on the
glass slide and observe under microscope.
VI. Rhizopus stolonifer (Fr.) Lind

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Rhizopus stolonifer causes most destructive disease of sweet potato in storage.

The pathogen affects many vegetables and fruits when conditions are favorable for
infection and disease development.
Morphological characters: Mycelium coenocytic with stolon and rhizoid,

sporangiophore bearing a terminal sporangium; columella spherical; ripe sporangiospores
mostly ovoid; gametangia similar in size and shape; zygospores covered with a black,
thick, several-layered wall.
Procedure
1. Examine a rotting sweet potato and note the coarse, whiskery growth; brownish,
soft and stringy fleshy tissues and a mild odour. Press the rotten area and watch
the water exuding through the broken skin.
2. Mount the whiskery growth and examine under the microscope. Note mycelium,
rhizoids, stolons, sporangiophores, columella, sporangia, sporangiospores,
gametangia and zygospores.
3. Take the small amount of the growth from the pathogen culture, mount it on the
glass slide and observe under microscope.
VII. Erysiphe polygoni DC
In India, the powdery mildew diseases are wide spread, which attack the plants in
all the stages of growth. Several leguminous crops are attacked by the pathogen.
Morphological characters: Mycelium superficial; haustoria present; conidiophores

septate, arising vertically from the superficial mycelium on the host surface bearing
conidia in chain; conidia elliptical, barrel-shaped or cylindrical, hyaline, unicellular;
cleistothecia black, minute, scattered in the mycelium; appendages mycelium-like; asci
ovate, sessile; ascospores elliptical, hyaline, unicellular.
Procedure
1. Notice white, powdery patches on the affected parts. Examine the scrape from
leaf under the microscope and note the morphology of conidiophores and conidia.
2. Examine the slide mounted with cleistothecia, asci and ascospores under the
microscope and note the" morphology of cleistothecia, appendages, asci and
ascospores.
VIII. Uncinula necator (Schw.) Burr

Uncinula necator is responsible for causing the powdery mildew in grapes. This
disease appeared in epidemic form in European countries during this mid-century period.
It is now known to occur in all the countries where grapes are grown and is serious
problem than the downy mildew in regions where relatively dry conditions prevail during
the growing season.

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Morphological characters: Mycelium grayish white, septate, superficial; conidiophores

short, club-shaped; conidia hyaline, ovate to oblong, nonseptate, borne in chains at the
apex of the conidiophores; cleistothecia black, globose with hooked appendages; asci
ovate; ascospores hyaline, oval, nonseptate.
Procedure
1. Examine the upper surface of leaves with powdery patches or dusty coating; canes
with similar patches and discolored, brown and black areas; withered flowers and
spotted and misshapen young berries.
2. Examine the powdery mass under the microscope and note conidiophores and
conidia.
3. Examine the slide mounted with cleistothecia, asci and asc6spores and note the
morphology of cleistothecium, appendage, ascus and ascospore.
IX. Sphaerotheca fuliginea (Schlecht) Paliacci

Sphaerotheca fuliginea is the main cause of powdery mildew of cucurbits in India.
Morphological characters: Mycelium is hyaline, occasionally brown when old, usually

evanescent but sometimes persistent. Conidia are formed in long chains, often with
distinct fibrosin bodies, ellipsoidal to barrel shaped, 25-37 x 14-25 µm in size.
Cleistothecia are scattered to densely gregarious, 66-98 µm in diameter, usually less than
85 µm with wall cells usually 25 µm wide. Appendages are usually variable in number, as
long as the ascocarp, myceloid, brown. Asci are broadly elliptic to subglobose, 50-80 x
37-60 µm in size. Each ascocarp contains a single ascus. There are 8 ascospores in the
ascus, they are ellipsoid to nearly spherical, 17-22 x 12-20 µm in size.
Procedure
1. Notice white, powdery patches on the affected parts. Examine the scrape from
leaf under the microscope and note the morphology of conidiophores and conidia.
2. Examine the slide mounted with cleistothecia, asci and ascospores under the
microscope and note the morphology of cleistothecia, appendages, asci and
ascospores.
X. Puccinia graminis f.sp. tritici Erikss. & E. Henn.
The Puccinia graminis f.sp. tritici is responsible for causing wheat rust in every
wheat growing area around the world. In Northern India, it appears late and does not
cause much damage, but in Southern and Peninsular India, it causes heavy losses because
of its early appearance.
Morphological characters: Uredospores brown, oval, echinulate; teliospores, chestnut

brown, bi-celled, apex pointed or rounded, attached firmly to stalk; pycnia flask-shaped;
pycniospore hyaline; aecial cups tube-like; aeciospores yellow, echinulate in chains.

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Procedure
1. Note oblong to circular, brick red, reddish brown and black pustules frequently
merging with one another on leaves and stem of wheat.
2. Examine brick red to reddish brown pustules (uredia) and dark brown to black
pustules (telia) under the microscope and note the ruptured epidermis.
3. Cut sections of the leaves and stem of wheat, stain and examine under the
microscope. Note the colour and shape of uredospores add teliospores. Notice
pedicel of teliospore.
XI. Sphacelotheca sorghi (Link) Clint.
Sphacelotheca sorghi is responsible for causing the grain smut of sorghum in all
the sorghum growing countries in the world. In India, it is more prevalent in Andhra
Pradesh, Maharashtra, Gujarat, Uttar Pradesh, Mysore, Tamil Nadu and Rajasthan.
Morphological characters: Sorus with a tough wall and a long, hard, central tissue
(columellum); teliospores, dark brown to black, smooth, thick-walled; promycelium 4
celled, producing a single sporidium from each cell or germ tubes instead of sporidia.
Procedure
1. Examine the diseased ear head and notice oval to cylindrical and dirty grey sori in
place of grains. Note the tough wall of sorus and the shape of columellum.
2. Mount teliospores in a drop of Lacto-phenol and examine under the microscope.
Note morphology of teliospores. Examine germinated teliospores and note
morphology of promycelium and sporidial germ tubes.
XII. Ustilago tritici (Pers.) Rostr.
Ustilago tritici is responsible for causing loose smut of wheat. The disease is
worldwide in its occurrence. In India, it is found on wheat in the plains as well as in the
hills.
Morphological Characters: Teliospores olivaceous brown, lighter on one side, spherical

or oval; epispore with fine spines especially on the lighter side; promycelium giving rise
to infection threads.
Procedure
1. Examine the diseased ear head and notice all the spikelets except the awns
transformed into a black powdery mass. Note that in some ear head only the
central axis is left behind.
2. Mount a small portion of the black powder in a drop of Lacto-phenol and examine
under the microscope. Note the morphology of teliospores. Examine the
germinated teliospores and note the formation of promycelium and infection
threads.

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XIII. Agaricus biosporus (Lange) Singer

This is the fungus used as the food. Mostly they are saprobes on soil or dead wood
Morphological characters: Basidiocarps are largely fleshy, membranous, pliant or
fragile hymenophore is poroid. The fruit is mostly centrally stipitate. The hymenophore is
rarely smooth to typically lamellate. It is gymnocarpic, pseudoangiocarpic or angiocarpic.
The basidia are 1 celled at maturity and produce 2 to 4 or even 8 basidiospores. The
spores are forcibly discharged from sterigmala.
Procedure
1. Examine the sample of mushroom given to you.
2. Cut the fine section of the specimen given to you and observe under the
microscope.
3. Observe the permanent slide of the basidium and basidiospore.
XIX. Pleurotus sajor caju (Fr.) Singer
This is the fungus used as the food. Mostly they are saprobes on soil or dead wood.
Morphological characters: Basidiocarps are largely fleshy, membranous, pliant or
fragile hymenophore is poroid. The fruit is mostly centrally stipitate. The hymenophore is
rarely smooth to typically lamellate. It is gymnocarpic, pseudoangiocarpic or angiocarpic.
The basidia are 1 celled at maturity and produce 2 to 4 or even 8 basidiospores. The
spores are forcibly discharged from sterigmala.
Procedure:
1. Examine the sample of mushroom given to you.
2. Cut the fine section of the specimen given to you and observe under the
microscope.
3. Observe the permanent slide of the basidium and basidiospore.
Record:Record the following observations

1. Symptoms of the disease.
2. Different structures of pathogen and microscopic sketch.
3. Classification of the pathogen.
***************

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EXERCISE No. 5
STAINING AND IDENTIFICATION OF
PLANT PATHOGENIC BACTERIA
Object : To study the staining and identification of plant pathogenic bacteria.
Methods : 1. Simple staining
2. Gram staining
3. Capsule staining
4. Flagella staining
5. Endospore staining
1. Simple staining :
Material: Bacterial culture, methylene blue or carbol fuchsin, slides, spirit lamp,
glass wash bottle, muslin cloth, microscope, slide holder.
Procedure:
1) Clean the slide with detergent powder to wash of greasy surface. Dry the slide in air.
2) Sterilize the slide over flame on both the slides.
3) Preparation of smear: A little drop of bacterial suspension is mixed in distilled water and
placed on the slide, spread it uniformly and thinly with a glass rod or needle to form a
very thin film of smear.
4) Drying: Allow the smear to dry in air only. Do not dry on flame. It forms clusters on
flame.
5) Fixing: Warm the slide slightly by passing through the flame two to three times so that
the bacteria get fixed on the slide.
6) Staining: Place two or three drops of any simple stain over the smear and allow to react
for specific time. (Methylene blue 1 to 1 ½ minutes, carbol fuchsin –5 to 10 seconds)
7) Washing: Wash the slide with gentle stream of water, wipe out the lower surface and dry
the upper surface in air.
8) Mounting: Observe under low power. Select a good spot. Bring it in the center, then
adjust under high power and finally observe under oil immersion objective.
Composition of Methylene blue (aqueous 3%)
Methelene blue 0.30 g
Ethenol (95%) 30.0 ml
Dissolve methylene blue in ethanol and then mix in distilled water.

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Class work:
1. Prepare a stained smear of bacteria.
2. Record the shape and cell groping of bacteria.
2. Gram staining :
Object : To study the type of differential staining which divides bacteria into
two groups namely (1) Gram + ve, 2) Gram – ve.
Principle: The organisms are first treated with main stains, and then subjected to decolouring
agent. Later on, they are counterstained with other dye. The organisms that retain the colour of
the main stain are called as ‘Gram positive’. Whereas, those bacteria, which loose the colour of
the main stain during deodorization & take up the colour of counter stain are called as a ‘Gram
negative’.
‘Christien Gram’ first used the stain in 1984 to demonstrate the proportion of bacteria in
the diseased tissue. There are different modifications of Gram staining using the same principle.
Material: Glass slide, 24 hrs. fresh bacterial culture, Gram A and Gram B solution, Gram’s
Iodine solution, 50% Acetone-Alcohol solution. Basic fuschin, spirit lamp, glass rod, wash bottle,
muslin cloth, slide holder, microscope.
Procedure: Kopeloff and Beerman’s method

1) Prepare a thin smear on slide, dry in air and fix on flame.
2) Cover the smear with gram ‘A’ and Gram ‘B’ solution in 3:1 proportion (6 drops of Gram
‘A’ and 2 drops of Gram ‘B’). Allow it to react for 5 minutes.
3) Treat with iodine solution (Mordant). This helps in fixing the colour of the main stain. It
is done by dipping the slide in a jar containing Iodine solution for 2 minutes.
4) Decolourization: Pass the slide, serially through three jars containing 50% acetone-
alcohol solution.
5) Wash with water and dry in air.
6) Counter stain with fuchsin for 20 to 30 seconds.
7) Wash the slide, dry in air and examine under microscope with high power.
Results:
If the bacteria retain colour of main stain and appear violet or blue, they are ‘Gram +ve
and if they take up the colour of counter stain showing red colour they are ‘Gram –ve’.
Definitions:
1. Mordant: It is a chemical agent which helps in fixing the colour of the main stain e.g.
Iodine solution.

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2. Decolourizing agent: It is a chemical that decolourises (removes) the main colour of the
main and thus the colour is lost from the cell.
3. Counter stain: It is stain used for staining after the main stain is decolourised.
Composition of stains
I) i) Main stain (A) : Crystal or Genstion violet – 1.0 g
Distilled water - 100 ml.
ii) Gram –B : Sodium carbonate - 1.0 g
Distilled water - 20 ml.
II) Counter Stain
i) Basic fuchsin : 1.0 g
ii) Distilled water : 100 ml
IV) Mordant
i) Iodine crystal : 2 g.
ii) NaOH (N/10) : 10 ml
Make Volume up to 100 ml.
Examples of Gram +ve and Gram –ve bacteria
Gram +ve Gram –ve
Bacillus subtilis, Bacillus anthracis, Lactobacillus Escherichia coli, Azotobacter

bulgaricus, Mycobcterium tuberculosis, chroococcum, Rhizobium
Streptomyces scabies, Corynebacterum leguminosarum, Xanthomonas citri,
diphtheriae, Clostridium tetani, Diplococcus
X. malvearum, Psedomonas
penmoniae, Mycobacterium lepreae, Streptococcus
solanacearum, Erwinia amylovora
lactis
Class work:
1) Prepare stained smear from dilute suspension of 24 hr old culture of
Xanthomonas citri or X. malvacearum and record gram reaction.
2) Draw a neat sketch of gram +ve and gram –ve cells.

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EXERCISE NO. 6
Study of Phanerogamic Plant Parasites
Most of the diseases are caused by fungi bacteria and viruses. There are few seeds
plants called flowering parasites (Phanerogams) which are parasitic on living plants.
Some of these attack roots of the host, while some parasites on stem. Some are devoid of
chlorophyll and entirely dependent on their host for food supply, while other have
chlorophyll and obtain only mineral constituents of food from host by drawing nutrition
and water they are called as Holoparasites or complete or total parasite. They have
haustoria as absorbing organs, which are sent deep into the vascular bundle of the host to
draw nutrients, water and minerals.
Flowering Plant Parasites: There are two types of parasites.

1) Root Parasites:
i) Striga (Partial root parasite)
ii) Orobanche (Complete root parasite)
2) Stem Parasites:
i) Dodder (Cuscuta) (Complete stem parasite)
ii) Loranthus (Partial Stem parasite)
1. Root Parasites:
1. Total or Complete or Holoparasite: Orobanche (Broom rape or Tokra)
It is annual flashy flowering plant growing to height of about 15-50 cm long,
yellow or brownish colour and covered by small thin scaly leaves. Flowers appears in the
axil of leaves are white or tubular. Fruits appears in the axil of leaves are white or tubular.
Fruits are capsule containing and seeds are very small, black in colour remain viable for
several years. The hausteria of parasite penetrates into the roots of hosts and draw its
nourishment. The growth of the plant is retarded, may die some times. It attacks tobacco,
tomato, brinjal, cabbage, cauliflower.
2. Hemi Partial or Semi Root Parasite: Striga (Witch Weed or Turfula or Talop)
Family : Scrophulariaceae
It is a small plant with bright green leaves grows upto height 20-60 cm leaves
beers chlorophylls and developed in clusters of 10-20 % host plant. They are obligate

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parasites therefore, do not obtain all their nutrient from their host root. Flowers are pink
in colour, seed are very minute and produce in grate number 5000 to 100000 seeds plant
per years. One flower contain 1200-1500 seeds and remains viable upto 12-40 years.
Dissemination takes place with rain water, flood, wind and irrigation water. It cause
yellowing and wilting of host leaves. It attacks sugarcane jowar, Maize, cereals and
millets.
b. Stem Parasites:
1. Total or Complete or Holoparasite: Cuscuta or dodder (Amarvel, Lovevine)
Family - Cuscutaceae.
Genus – Cuscuta
It is non chlorophyllous, leaf less parasitic seed plant. It is yellow pink or orange
in colour and attached to the host. They do not bear leaves but bear minute function less
scale leaves is produces flower and fruits. Flower are white, pink or yellowish in colour
and found in clusters. Seed are form in capsules. A single plant may be produce 3000
seeds.
The first appearances of parasites is noticed as thread like leaf less stem which
devoid of green pigment and twine around the stem or leaves of the host. When stem of
parasitic plant comes in contact with host, the minute root like organs. i.e. haustoria
penetrates into the host and absorbs. When the relationship of the host is firmly
established, the dodder plant looses the contact from soil.
These affect plant get weakened and yield poorly the seeds spread by animals,
water and implements and remain viable when condition are unfavorable.
It attacks berseem alfalfa, clover, flax, onion, potato, ornamental and hedge plants.
2. Partial, Semi or Hemi Stem Parasites:a

Loranthus
Family- Loranthaceae.
It is a partial parasite of tree trunks and branches with brown stem, dark green
leaves but no roots.
1. Stem is thick and flattened of the node, appear in clusters at the point of attack which
can be easily spotted on the trees.
2. At the point of attachment with the tree, it shows swellings or tumorous growth where
the haustoria are produced. It produces flowers which are long, tabular, greenish,
white or red colour and found in clusters. It produces fleshy berries with single seed.
3. The affected host plant becomes stunted in growth and dispersal of seed is mostly
through the birds and animals. It attacks mango, citrus, apple, guava.

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Exercise no. 7
Transmission of Plant Viruses
Plant viruses are obligate parasites, often causing the death of their host, so it is
necessary for them to spread from plant to plant and to be introduced into living cells.
The knowledge of virus transmission is important to: Recognize a virus as cause of the
disease if transmitted from infected to healthy plant How virus spread in field – help in
its control Establish biological relationship of interaction between virus and its vector
Mechanical transmission is very important for lab study of viruses
There are two types of plant virus transmission :
Horizontal transmission: Horizontal transmission is by vectors, human, pruning shears

and tools and other direct external contamination.
Vertical transmission: Vertical transmission occurs when a plant gets it from its parent
plant. Either through asexual propagation (cuttings) or in sexual reproduction via infected
seeds.
This spread or transmission will be considered under the following headings:
1. Mechanical transmission
2. Vegetative, graft and dodder transmission
3. Transmission by pollen and seeds
4. Insect and mite transmission
5. Nematode and fungal transmission.
Mechanical transmission
Methods
Leaf rub
Cotton swab
Pinprick
Microinjection
Steps: Sap extraction, Extraction medium, Use of additives, Choice of suitable host:
most common are Nicotiana spp., Chenopodium spp., Cucumis sativus, Gomphrena,
Datura spp, Phaseolus vulgaris.

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Exercise No. 8
STUDY OF MORPHOLOGY FEATURES AND
IDENTIFICATION OF PLANT PARASITIC NEMATODES
Objectives
 To distinguish plant parasitic and free living nematodes.

 To differentiate males, females and juveniles.
 To study important morphological features of body wall, alimentary canal and
reproductive systems of plant parasitic nematodes.
Observations
Examine the nematodes under various magnifications of a compound microscope/

stereomicroscope and note the following characters.
A. Toto mount (Fig. 14 )
 Body vermiform, with clear transparent anterior region (oesophageal region).

 Openings of digestive system (anus), excretory system (excretory pore) and
reproductive system (vulval/cloaca! opening) on ventral sides.
 Identify dorsal and lateral sides.
B. Digestive system (Fig. 15)
 Made up of a stoma provided with a knobbed stylet, oesophagus, cardia, intestine

and rectum.
 Stylet : Made up of a conns, shaft and three knobs.
 Oesophagus : Consisting of an anterior slender part (procorpus), median bulb with
valvular apparatus, isthmus and a basal bulb. Locate the position of oesophageal
gland in the basal bulb and the opening of dorsal oesophageal glands behind the
stvlet knobs.
<<
C. Female reproductive system (Fig. (14 )
 Number of genital branches and classify into didelphic/ monodelphic.

 Orientation of genital branch(es) in relation to vulva and classify as prodelphic /
opisthodelphic / amphidelphic.
 Presence of sperm in spermatheca.
 Location of vulva.

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D. Male reproductive system (Figs, 14 & 15)

 Number of testes : Monorchic/diorchic and their orientation.
 Common opening of reproductive and digestive system. Cloaca.
 Number and shape of spicules.
 Location of gubernaculum.
 Caudal alae (bursa).
E. Difference between female, male and juvenile
Female Male Juvenile
1. Independent opening of Common opening of No opening of

reproductive system (Vulva), digestive and reproductive reproductive system
and digestive system (anus) systems (cloacal opening )
near the posterior end
2. Posterior region of body Spicule, gubernaculums and As in female

without spicules, gubemaculum caudal alae present
or caudal alae
F. Examine a live nematode suspension and note the nematode movements. Distinguish
plant parasitic nematode from saprophylic by the presence of a knobbed stylet.
Tylenchid oesophagus (made up of procorpus, valvcd median bulb, isthmus
and non-valvate basal bulb).
G. Cross section of nematode body (Fig. 15 )

Rounded in shape; body wall made up of cuticle, hypodermis and somatic
musculature; hypodermal chords; intestine; pseudocoelome containing parts of
reproductive systems.
*******

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EXERCISE NO. 9
PREPARATION OF MEDIA
Plant pathogens such as fungi and bacteria can be cultivated in vitro on various
media, which can be either solid or liquid. For solidifying any media, a jellifying material
generally agar-agar is added into it or sometimes it is so because of the nature of the
media itself Due to great variety of the nutrient requirement of different pathogens, the
composition of the media used for their cultivation varies.
Based on the composition, media can be differentiated as natural, semi synthetic

and synthetic ones.
1. Natural media
Natural media are most favorable to growth and sporulation than synthetic ones.
The fungi, in general, grow best on media containing natural, basic materials like plant
extracts such as decoctions, extracts, juices from host plant parts or pieces of host tissues
added to water agar or placed on sterile moist sand or water or yeast extracts. Propylene
sterilized fruits, leaves, stems, straw, and roots of the host and some times non-host plants
are excellent substrates for fructification in fungi. The ingredients used vary from time to
time as the quality of the medium depends upon the state of the substance Therefore; it is
not suitable to conduct precise metabolic investigation on natural media because of
variability in the results.
Few examples of commonly used natural media are, Potato plug, lupine stem,
prune juice agar, peanut leaf-oat meal agar, carrot agar, carrot juice agar, rape seed agar,
rice grain, pea seed medium, hernp seed, agar, bean juice agar, corn/maize meal agar,
apple juice agar, apple leaf decoction agar, oat meal agar.. malt-extract agar, potato
dextrose agar, etc.
2. Semi synthetic media
Semi synthetic media are those in which all but one or two ingredients can be
duplicated for example Malt-Czepak's agar, Yeast extract-dextrose agar, Leonian solution
agar.
3. Synthetic media
Synthetic media are those whose chemical composition is known precisely. They
are costly and take more time to prepare than natural media. The choice depends upon the
put-pose and the requirements of the experiment. No universal rules or recommendations
can be outlined for the synthetic medium suitable for sporulation of all fungi. Few
examples are Czapek's dox agar, Peptone-dextrose agar, Peptone rose bengal agar, etc.

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4. Selective media
It is possible to isolate specific plant pathogens by using selective media

irrespective to the category of the medium. Suitable selective medium has to be identified
for different pathogens. Nutrient agar (containing 5% saccharose (NASA) + 2ppm crystal
violet) and King's agar B media are found most suitable for isolating and detecting
Pseudomonas syringae pv. phaseolicola in Phaseolous vulgaris seed.
PREPARATION OF MEDIA
Most of the media can be routinely prepared easily in the following way.
• It is preferable to use a weak medium’ with a reduced carbon and/or nitrogen

source to induce sporulation and suppress the vegetative growth.
• The ingredients, amounts as indicated in the formula or recipe, are added to a

flask that should hold 1-1.5 or 2 times the final volume of medium.
• Measure the required volume of water (de-mineralized or distilled water) and add
the ingredients, little by little with constant stirring to wet the ingredients
thoroughly and to avoid the formation of lumps.
• Cover the flask with aluminum foil or plug the neck with cotton.
• Heat the flask to dissolve the ingredients (a microwave oven can be used, but
make sure that the flask is not too tightly closed). If agar is a component of the
medium, heat the mixture to liquify the agar.
• Sometimes checking of pH is required. The reaction of the medium can be

determined by pH indicator papers, or by a pH meter. The reaction is adjusted to
the desired level by slowly adding drops of IN NaOH or HCI to the hot solution.
• The medium while still hot is dispensed to appropriate containers for sterilization.
If the medium is to be used for propagation of a bacterial pathogen, it is usually
dispensed into test tubes .
 The medium should be sterilized the same day as it is prepared. Sterilization is

done in a steam autoclave . Tubes are loosely arranged in wire basket or other
containers. Sterilization is completed in 20 minutes at a temperature of 1210C.
 Media containing sugars, vitamins and growth substances should better be
sterilized by fractionally steaming at 100'C for 2-3 consecutive days. After the
required time elapsed, the pressure should be allowed to fall gradually to zero
before the medium is removed from the sterilizer.

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Precautions
1. Rich carbohydrate medium helps to

get profuse mycelial growth but reduces sporulation.
2. Fungi usually prefer slight acidic
medium (pH 6.0-6.5) while bacteria prefer pH 7.
3. Agar does not properly solidify in
very acidic or alkaline media.
4. Tap water is often preferable to
distilled water as it has useful trace elements. However in some areas it may be toxic
and Phytopthora species are particularly sensitive and in that condition glass distilled
water is better to be used.
5. Over sterilization is harmful as it
decomposes proteins and carbohydrates present in the medium.
PREPARATION OF SLANTS
For preparing the slants, medium is poured in culture tubes. Approximately 1/4 part of the
tube is filled in and plugged with cotton plugs. After sterilization, tubes are tilted to
450-600angle till medium solidifies properly. Care is taken that the medium should not
touch the inserted cotton plugs in the tubes.
PREPARATION OF COTTON PLUGS
Culture tubes or flasks are plugged before sterilization with cotton plugs until or
stated. Following precautions are required while preparing the cotton plugs.
 Use non-absorbent cotton

 Cotton should be tightly rolled to avoid loosening of the plugs.
 Plugs may be used as such or they may be wrapped with muslin cloth
 Plugs neither should be very tight nor loose in flasks or tubes.
 Plugs may be reused
*****

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EXERCISE NO. 10
ISOLATION AND PURIFICATION OF FUNGI AND BACTERIA.
ISOLATION:
It is a technique of separating the organisms from the diseased host tissue and
growing them on artificial media for the purpose of further study or investigation.
METHODS OF ISOLATION:
A) FUNGAL ISOLATION
Isolation of fungal plant pathogens from diseased host tissue. When causal
organism is located inside the host tissues.
ISOLATION FROM DISEASED HOST TISSUE:
1. Wash the diseased part with tap water to remove dirt and soil and cut the affected
part in to small convenient pieces of 2 to 3 mm size in such a way that half the
portion of healthy tissue must be present on bits.
2. Surface sterilization with HgCl2 (Mercury chlorides) solution (0.1%) by
immersing the pieces for 2 to 5 minutes depending upon the type of tissue.
3. Rinse the pieces of the diseased bits in distilled sterilized water, for three or four
times so as to remove the traces of disinfectant i.e. HgCl2.
4. Place the pieces of bits on slants or Petri plates with pre poured potato dextrose
agar medium.
5. Incubate the Petri plates in inverted position at optimum temperature 25-30 0C for
7 days
6. After incubation period there is growth on the diseased bits, transfer the growth of
organisms in new slants .Purify the culture either hyphal tip isolation or by single
spore isolation method.
NOTE : If the infection is due to fungus, few drops of 2% lactic acid are mixed in the
sterilized water, in the third change to avoid bacterial contamination or add the antibictics
like penicillium and streptomycin @ 100 ppm/lit. of medium in order to avoid the
bacterial contamination.

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B) BACTERIAL ISOLATION:
1. Collect the samples suspected to be of infected with the bacteria.
2. Wash the sample in the running water, and dip them in sodium hypo chloride
solution (70%) for surface sterilization for few minutes.
3. Place the diseased tissue in a drop of sterilized water and cut into small pieces
with sterilized scalpel. The bacteria will ooze out in about 5 minutes.
METHOD – I:
1. Take a loopful of bacterial suspension and streak it on Petri plate containing the
nutrient agar Medium and incubate it in incubation at a temp. of 25 ± 2 0C on 72 to
96 hrs.
2. After 72 hours if colonies appear along the streak. Collect such a single
colony and transfer it on nutrient agar slant.
METHOD – II
1. Dilute the bacterial suspension with the help of sterilized distilled water. Add the
suspension to the liquefied medium when the temp. is approximately 40 0C and
mix thoroughly by shaking the medium and pour in sterilized Petri plates.
2. Incubate this Petri dish in inverted position in order to avoid contamination in an
incubator at 25 ± 20C for 72 to 96 hours.
3. After 72 hours if colonies appear on the medium, collect a single colony and
transfer it on the nutrient agar slant.
PURIFICATION OF FUNGAL CULTURE:
Following two methods does the purification of the fungal culture.
A. HYPHAL TIP ISOLATION METHOD:
In this method initially the growing hyphal tip is selected and transferred in the slant
to obtain the pure culture.
1. Grow the colony of the fungus on the PDA medium.

2. Select the single growing hyphal tip of the mycelium with the help of microscope
and mark it with glass marking pencil.

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3. Cut the marked portion with the help of cork borer and transfer it on the PDA
slant medium and incubate at the temp. 27 + 10C and observe for pure growth of
culture.
B. SINGLE SPORE ISOLATION METHOD :
1. Prepare the spore suspension from the sporulating fungus in order to have 10 6
spore/ml.
2. Add above 10 ml of spore suspension in 250 ml of liquefied potato destroe agar
medium when the temp. of medium is around 400C.
3. Add the medium in sterilized Petri dish, allow to solidity the medium,
4. After the solidification of the medium locate the single spore with the help of
microscope and mark with a glass marking pencil.
5. Cut the marked portion with the help of cork borer and transfer it on the PDA
slant.
6. Incubate the slants at the temp. 27 ± 20C and observe for pure growth of culture.
PURIFICATION OF BACTERIAL CULTURE :
STREAK METHOD:
The bacterial cultures are generally purified by mean of streak method. In this
method the loopful contaminated bacterial culture is streaked on the consecutively on the
three to four pre pore Petri plate containing nutrient agar medium. Then the plates are
incubated at 27 ± 20C for 72 to 96 hours. The isolate colonies from the last streaked plate
is generally picked up and to obtain pure culture. Select the isolated colony and streaked
it on slant containing N.A. medium.
Exercise:
1 Isolate the pathogen from the diseased sample provided to you ?

2 Note down morphological characteristics of the colonies of organisms growing on
artificial media ?
3 What is the mode of infection of grain smut of jowar and cotton wilt ?
4 What is incubation period?
5 What are the factors responsible for development leaf spot of cotton and fruit rot
of lemon?

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EXERCISE NO. 11
EXTRACTION OF NEMATODES FROM SOIL SAMPLES
Objective : To extract various types of nematodes from a soil sample for qualitative and
quantitative estimation.
Materials required : Beakers, enamelled metallic pans, petri-plates, sieves, soil sample,
facial tissue papers, wire nets, glass funnels, funnel stand.rubber tube, glass
vials.
Methods :
1. Cobb's decanting and sieving technique
Principle
 The soil particles and nematodes settle at different rates due to differences in their
specific gravity.
 Different sized nematodes are retained on sieves of different pore sizes.
Procedure (Fig. 2.1)
 Take out the composite sample in an enamelled metallic pan, mix it thoroughly
and take 250 cc for processing. Store the remaining sample in a refrigerator.
 Transfer 250 cc soil to Pan A and add about one litre water, mix well breaking
clods and clumps.
 Wait for 10-20 seconds and pass this soil suspension through a 20-rnesh sieve
(pore size 840jji.ni), collecting the filterate in pan B. Wash the pan A.
 Collect roots present on the 20-mesh sieve in a beaker and discard the
remaining material.
 Stir the suspension of pan B gently, wait for a few seconds and pour it through a
60-mesh sieve (pore size 250 nmin pan A. Collect the residue left over 60-mesh
sieve in a beaker and lable it as 60.
 Pass the contents of pan A through a 300-mesh sieve (pore size 53 m).
Discard the suspension passed through the sieve.
 Collect the residue left over 60-mesh sieve in a beaker and label it as 300
 Examine the contents of beakers labelled as 60 for cyst nematodes directly under
a steromicroscope.
 Further process 300-mesh residue by the following techniques.
2. Baermann's Funnel Technique Principle

 The active and motile nematodes pass through the tissue paper and get
collected at the base of rubber tube/specimen vial due to movement and

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gravitational force whereas inert soil particles/debris remain on the tissue paper.
Procedure (Fig. 2.2)
 Process the soil sample by Cobb's decanting and sieving technique as above.
 Take a glass funnel with a piece of rubbertubsr (10-12"long) bearing a glass vial
(5 ml capacity) attached to its distal end.
 Fill the funnel assembly with water and press the rubber tube gently to remove the
air bubbles.
 Transfer the sieved nematode suspension (labelled as 300) to a moulded piece
of wire net covered with a double layered tissue paper and place it over the funnel.
 Add water till it touches the lower surface of the wire net.
 After 24-48 hours remove the glass vial and observe the nematodes under a
stereoscopic binocular microscope.
Advantages
 Clear nematode suspension, free of debris etc. is obtained.

 The nematodes are collected in a small amount of water.
Disadvantages
 It is a time consuming.
 Nematodes may lose their activity/viability due to lack of oxygen.
 Sedentary and slow moving nematodes can not be extracted.
3. Modified Baermann's funnel Technique (Fig. 2,2)

In this technique petri-plate is used in place of funnel. Other steps arc same
as described above for Baermann's funnel method and the nematodes get
collected in petri-plate.
Advantage
Maximum recovery of active nematodes is achieved by this method the

because nematodes do not lose their activity/viability since the oxygen is
always available.
EXERCISE NO. 12

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KOCH’S POSTULATE
KOCH’S POSTIULATE
The main aim of Koch’s postulate to determine or establish the host pathogen
relationship between the host and a new micro-organism which is suspected to be
associated with the diseases.
Robert Koch studying with the anthrax disease in cattle followed out certain
conditions to prove the host pathogen relationship. These principle latter recognised as
the koch’s postulate.
KOCH’S POSTULATES
1) Isolation
When the organism is associated with the disease, it is separated i.e. isolated from
its natural host and grown under artificial conditions.
2) Inoculation
Introduction of isolated organism into healthy tissues. The individuals should

produce similar disease symptoms.
3) Reproduction of typical symptoms as in original
4) Re isolation
Re isolation of the inoculated organism (original organism) from artificially

diseased individuals. The re isolated organism should be similar to the original organism
inoculated.

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EXERCISE ON. 13
STUDY OF FUNGICIDES AND THEIR FORMULATIONS
Object: To study different groups of fungicides, their classification and diseases

Controlled.
WHAT IS FUNGICIDE?
The word ‘fungicide’ originated from two Latin words, viz., ‘fungus’ and ‘caedo’.
The word ‘caedo’ means ‘to kill.’ Thus, the fungicide is any agency/chemical, which
has the ability to kill the fungus. According to this meaning, physical agents like ultra
violet light and heat should also be considered as fungicide. However, in common
usage, the meaning is restricted to chemicals only. Hence, fungicide is a chemical that
is capable of killing fungi.
Fungistat
Some chemicals do not kill the fungal pathogens. However, they simply arrest the
growth of the fungus temporarily. These chemicals are called fungistat and the
phenomenon of temporarily inhibiting the fungal growth is termed as fungistasis.
Antisporulant
Some other chemicals may inhibit the spore production without affecting the
growth of vegetative hyphae and are called as ‘Antisporulant’ The antisporulant and
fungistatic compounds do not kill the fungi
Though, the fugistats and antisporulants do not kill fungi, they are included under
the broad term fungicide. Hence, in broad sense, the fungicide is defined as “A
chemical substance which has the ability to prevent damage caused by fungi to growing
crops and their products”.
Fungicides can be broadly grouped based on their
(i) mode of action (ii) general use and (iii) chemical composition.
I. Mode of action
Protectant: As the name suggests, protectant fungicides are prophylactic in their
behaviour. Fungicide, which is effective only if applied prior to fungal infection, is
called a protectant, e.g., zineb, sulphur.
Therapeutant: Fungicide that is capable of eradicating a fungus after it has caused
infection and thereby curing the plant is called ‘chemotherapeutant’, e.g. carboxin,
oxycarboxin antibiotics like Aureofungin. Usually chemotherapeutants are systemic
in their action and affect the deep-seated infection.
Eradicants: Eradicants are those which remove pathogenic fungi from an infection
court (area of the host around a propagating unit of a fungus in which infection could
possibly occur), e.g., organic mercurials, lime sulphur, dodine, etc. These chemicals

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eradicate the dormant or active pathogen from the host. They can remain effective on
or in the host for some time.
II. Based on general uses

The fungicides can also be classified based on their use in managing the diseases.
1. Seed protectants: E.g., captan, thiram, organomercuries, carbendazim, carboxin,
etc.
2. Soil fungicides (preplant): E.g., chloropicrin, formaldehyde, vapam, dazomet, etc.
3. Soil fungicides (for growing plants): E.g., Bordeaux mixture, copper oxychloride,
captan, PCNB, thiram, etc.
4. Foliage and blossom protectants: E.g., capton, ferbam, zineb, mancozeb,
chlorothalonil, etc.
5. Fruit protectants : E.g., captan, maneb, carbendazim, mancozeb, etc.
6. Eradicants: E.g., organomercurials, lime sulphur, etc.
7. Tree wound dressers : Eg. Boreaux paste, Chaubattia paste, etc.
8. Antibiotics: E.g., Actidione, Griseofulvin, Streptomycin, Streptocycline, etc.,
9. General purpose sprays and dust formulations.
III. Based on chemical composition
Non-systemic fungicides
i. Sulphur fungicides
Use of sulphur in plant disease control is probably the oldest one and can
be classified as inorganic sulphur and organic sulphur. Inorganic sulphur is used in the
form of elemental sulphur or as lime sulphur. Elemental sulphur can be either used as
dust or Wettable sulphur, later being more widely used in plant disease control. Sulphur
fungicides emit sufficient vapour to prevent the growth of the fungal spores at a distance
from the area of deposition. This is an added advantage in sulphur fungicides as
compared to other fungi toxicants.
Sulphur is a contact and protective fungicide, normally applied as sprays
or as dust. It is generally used to control powdery mildews of fruits, vegetables, flowers
and tobacco. This is also effective against apple scab (Venturia inaequallis) and certain
rusts, leaf blights of field crops and fruit diseases.
Dust formulations : Sulphur dust (300 mesh)
Lime Sulphur : It can be prepared by boiling 9 kg or rock lime and
6.75 kg of sulphur in 225 litres of water.
Wttable sulphur : Wetasul, Microsul, Thiovit, Sultaf, Cosan, Wetsulf,
formulations Sulfex, Cosavet, etc.
ii. Dithiocarbamates
Organic compounds of sulphur are now widely used in these days. All these
compounds, called as ‘carbamate fungicides’, which are derivatives of dithiocarbamic

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acid. Dithiocarbamates are broadly grouped into two, based on the mechanism of
action.
Monoalkyl Dithiocarbamates: E.g., zineb (Hexathane 75% WP, Dithane Z-78,
Funjeb, Lonocol, Parzate C), maneb (Dithane M22, Manzate WP, MEB), mancozeb
(Dithane M - 45, Indofil M - 45, Manzeb), nabam (Chembam, Dithane A-40, Dithane
D-14, Parzate Liquid), and vapam (Vapam, VPM, Chemvape, 4-S Karbation, Vita
Fume).
Dialkyl Dithiocarbamates: E.g., thiram (Thiride 75 WDP, Thiride 750, Thiram 75%
WDP, Hexathir, Normerson, Panoram 75, Thiram, TMTD, Arasan, Tersan 75,
Thylate, Pomarsol, Thiuram), ziram (Cuman L. Ziram, Ziride 80 WDP, Hexaazir 80%
WP, Corozate, Fukiazsin, Karbam white, Milbam, Vancide 51Z, Zerlate, Ziram,
Ziberk, Zitox 80% WDP), ferbam (Coromat, Febam, Ferberk, Femate, Fermate D,
Fermicide, Hexaferb 75% WP, Karbam Black, Ferradow).
Diseases controlled: These fungicides are used for control of a wide range of
diseases like leaf spots, blights, anthracnose, rusts, downy mildews, etc. of many
crops plants.
iii. Copper fungicides
The fungicidal action of copper was mentioned as early as 1807 by Prevost
against wheat bunt disease (Tilletia caries), but its large-scale use as a fungicide
started in 1885 after the discovery of Bordeaux mixture by Millardet in France. The
mixture of copper sulphate and lime was effective in controlling downy mildew of
grapevine caused by Plasmopara viticola and later, late blight of potato
(Phytophthora infestans).
Some other copper sulphate preparations later developed were Bordeaux paste,
Chaubattia paste, Burgandy mixture and Cheshunt compound, which are all very
effectively used in the control of several plant diseases. In addition, some
preparations of copper like copper oxychloride (Blitox 50, Cupramar 50 WP, Fytolan,
Micop D-06, Micop W -50, Blue copper 50, Cupravit, Cobox, Copper bond, Copter,
Cuprasol, Canopy, Cuprax, Bilmix 4% dust, Mycop, Topgun 50 WG), cuprous oxide
(Fungimar, Perenox, Copper Sandoz, Copper 4% dust, Perecot, Cuproxid, Kirti
copper) and copper hydroxide (Coside 101 – 77 WP) are also used.
Some of the important diseases controlled by copper fungicides:
Copper fungicides are used as protective fungicides for foliage applications and as
a soil drench. The freshly prepared BM has high tenacity. Diseases like late blight
(Phytophthora infestans) of potato, apples scab (Venturia inaequalis), downy mildew
of grapes, damping off of seedlings, pink diseases, stem canker, leaf diseases and
rusts of several crops, soil borne diseases viz., wilt, collar rot, root rots, etc. are
effectively managed by these fungicides. They are also used as wound dressers.
iv. Mercury fungicides
Mercury fungicides can be grouped as inorganic and organic mercury compounds.
Both the groups are highly fungitoxic and were extensively used as seed treatment
chemicals against seed borne diseases. These compounds show bactericidal property

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also. However, due to their residual toxicity in soil and plants and their extreme toxic
nature to animal and human beings, the use of mercury fungicides is beings
discouraged. In most of the countries, the use of mercury fungicides is banned and in
countries like India, the use of mercury fungicides is restricted only in seed treatment
for certain crops.
Inorganic mercury compounds: These are mercuric chloride (Merfusan, Mersil),
and mercurous chloride (Cyclosan, M-C Turf fungicide).
Organomercurials: Methoxy ethyl mercury chloride [Agallol, Aretan, Emisan,
Ceresan wet (India)], phenyl mercury chloride [Ceresan dry (India), Ceresol,
Leytosan], ethyl mercury chloride [Ceresan (USA)].
Diseases controlled: The mercury fungicides are used mainly for treatment of seeds
and planting materials, either by dry, wet or slurry method. For seed treatment, 1 per
cent metallic mercury is applied at 0.25 per cent concentration. Mercurous chloride is
limited to soil application in crop protection because of its phytotoxicity.
v. Heterocyclic nitrogen compounds
Heterocyclic nitrogen compounds are mostly used as foliage and fruit protectants.
Some compounds are very effectively used as seed dressers. Some of the commonly
used fungicides are captan (Captaf 50 WP, Cohocap 50WP, Captan 75 WP, Esso
Fungicide 406, Orthocide 406, Vancide 89, Deltan, Metpan, Hexacap), captafol (Foltaf,
Difolaton, Difosan, Captaspor, Foleid, Sanspor), glyodin (Glyoxaliadine, Glyoxide,
Glyodin, Glyoxide dry, Glyodex 30% liquid and 70% WP) and folpet (Phartan,
Acryptan, Phaltan, Folpan, Orthophaltan).
Diseases controlled: Captan and captafol are mainly seed dressing fungicides used to
control diseases of many seed and soil borne diseases of fruits, ornamental and
vegetables. In addition, captan, glyodin and folpet are used as foliar sprays for
management of diseases like leaf spots, blights, anthracnose, rusts, downy mildews,
etc. of many crops plants.
vi. Dicarboximide compounds
Iprodione (Rovral) and vinclozolin (Ronilan, Ornalin) are two important
fungicides in this group. They are broad spectrum, contact fungicides and control the
diseases caused by species of Botrytis, Sclerotinia, Monilinia, Alternaria,
Helminthosporium, Fusarium, Penicillium, Rhizoctonia, etc.
vii. Organo – Phosphorous fungicides
Ediphenphos is available as Hinosan 50% EC and 2% D. It has a specific action
against Pyricularia oryzae (blast), Corticium sesakii and Cochliobolus miyabeanus in
rice.
Systemic fungicides
Since the late 1960s, there has been substantial development in systemic
fungicides. Any compound capable of being freely translocated after penetrating the plant
is called systemic. A systemic fungicide is defined as fungitoxic compound that controls a
fungal pathogen remote from the point of application, and that can be detected and

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identified. Thus, a systemic fungicide could eradicate established infection and protect
the new parts of the plant. Based on chemical structure, systemic fungicides can be
classified as Benzimidazoles, Thiophanates, Oxathilins and related compounds,
pyrimidines, morpholines, organo-phosphorus compounds and miscellaneous group.
i. Oxathilin and related compounds
This group of systemic fungicide is also called as carboxamides, carboxylic acid
anillides, carboxa anillides or simply as anillides which are effective only against the
fungi belong to Basidiomycota and Rhizoctonia solani. Some of the chemicals
developed are Carboxin (Vitavax 10% D, Vitavax 75% WP, Vitavax 34% liq.
Vitaflow), and oxycarboxin (Plantvax 5G, Plantvax 5% liq. Plantvax 1.5 EC, 10%
dust, 75 WP).
Diseases controlled: These fungicides are used for the management of seed borne
diseases in cereals, pulses, ornamentals, vegetables, etc.
ii. Benzimidazoles
The chemicals of this group show a very broad-spectrum activity against a variety of
fungi. However, they are not effective against bacteria as well as fungi belonging to
phylum Oomycota. Two types of fungicidal derivates of benzimidazoles are
thiabendazole and fuberidazole. The fungicides in this group are carbendazim
(Bavistin 50 WP, MBC, Dersol, Sten 50, Zoom, Tagstin, Agrozim, Jkenstin), and
benomyl (Benlate 50 WP, Benomyl).
Diseases controlled: Effective against a wide range of fungi affecting field crops,
fruits and ornamentals. These fungicides are very effective against rice blast, apple
scab, powdery mildew of cereals, rose, curcurbits and apple, sigatoka leaf spot of
banana, turmeric leaf spot, rust diseases in many crops and diseases caused by
Verticillium and Rhizoctonia. They are also used for seedling dip, seed treatment, and
soil drench and as pre-and post-harvest sprays or dips for the control of storage rots of
fruits and vegetables.
iii. Thiophanates
These compounds represent a new group of systemic fungicides based on thiourea.
They are the derivatives of thioallophanic acid. These fungicides contain aromatic
nucleus, which is converted into benzimidazole ring for their activity. Hence,
thiophanates are often classified under benzimidazole group and the biological
activity of thiophanates resembles of benomyl. The important compound developed
under this group is thiophanate methyl (Topsin M 70 WP, Cercobin-M 70 WP,
Envovit-methyl, Mildothane).
Diseases controlled: Same as that of benzimidazoles.
iv. Morpholines
This group includes tridemorph (Calixin 75 EC, Bardew, Beacon). It controls
powdery mildew diseases of cereals, vegetables and ornamentals. In addition, it is
highly effective against Mycosphaerala musicola, Exobasidium vexans and rust
diseases or cereals, pulses and coffee.

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v. Pyrimidines
Pyrimidins include fenarimol (Rubigan), etc. It is very effective against powdery
mildews and rusts of several crops, Venturia and Monilinia in fruits (pome fruits).
vi. Triazole compounds
This group includes fungicides viz., triadimefon (Bayleton), bitertanal (Baycor),
bitertanol (Baycor), tricyclazole (Beam, Bim), hexaconazole (Contaf 5 EC,
ANVIL 5 EC), myclobutanil (Systhane), propiconazole (Tilt 25 EC), penconazole
(Topas 10 EC), difenoconazole (Score 25% EC), etc.
Diseases controlled: They are having broad-spectrum activity against diseases
like rust, powdery mildews, leaf spots, anthracnoses, etc. of several crops.
vii. Organo phosphorous compounds
It includes kitazin / iprobenphos (Kitazin 48% EC, Blataf). It is used to control
blast (Pyricularia oryzae) and sheath blight of rice.
viii. Piperazine - Triforine (Saprol-EG, Fungitex). Effective against powdery mildew,
scab, and rust on ornamentals and cereals and active against storage diseases of
fruits.
ix. Other systemic fungicides
Other systemic fungicides are metalaxyl (Apron 35 SD, Ridomil, Ridomil MZ 72
WP, Ridomil Gold), fosetyl AI (Alliette 80 WP), and cymoxanil (Curzate). They
are having very effective systemic selective action against diseases caused by
Oomycetes, e.g., damping-off, root rots, stem rots (Pythium, Phytopthora spp.),
and downy mildew of several crops.
Other important fungicides
The important fungicide, which is not included in any of the group described
earlier, is chlorothalonil (Kavach 25% WP, Bravo, Daconil, Termil, Chlorothalonil
40 SC, Safegaurd, Spektrum). It is broad spectrum, contact fungicide effective
against many fungal diseases of field, orchard, ornamental and plantation crops.
Miscellaneous fungicides
Many aromatic compounds have important anti-microbial properties and have been
developed as fungicides. Some important benzene compounds commonly used in
plant disease control are Quintozene, i.e., PCNB (Brassicol, Terraclor, Tritisan
10%, 20%, 40% D and 75% WP, PCNB 75% WP), and Dinocap (Karathane,
Arathane, Mildex, Crotothane).
Diseases controlled: PCNB is used for seed and soil treatment against Botrytis,
Sclerotium, Rhizoctonia and Sclerotinia spp. Dichloran is a protective fungicide
and very effective against Botrytis, Rhizopus and Sclerotinia spp. Dinocap is a non-
systemic acaricide and recommended to control powdery mildews on various
crops.
Note: The details of diseases controlled is the additional information for the
students though it is not included in syllabus.

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FUNGICIDE FORMULATIONS
The trade names under which different companies market various formulations of
the same compound vary widely. However, all marketed fungicides should state clearly
the common name of the fungicide and the amount of active ingredient of it contained in
the formulated product. Commercial fungicides are formulated in various ways and most
commonly available formulations are Emulsifiable Concentrates (EC) Wettable Powders
(WP), Dusts (D), etc.
Commercially available fungicides usually consist of a mixture of active
ingredient (a.i.) and other substances including diluents, wetting agents, stickers,
emulsifiers, etc. Formulations containing mixtures of different active ingredients
(especially mixtures of protectant and systemic fungicides) are also widely used now a
day. Different formulations incorporating the same active ingredient may be used for
distinct purposes like seed treatment, foliar application, etc.
Emulsifiable concentrates (EC)
These are liquid formulations which can be diluted with water before application. The
active ingredient is dissolved in a solvent. The fungicides and solvents will often not mix
with water, so an emulsifying agent or water dispersible oil is mixed. When these
emulsifiable concentrate is added to water, a milky mixture is formed, which is a
suspension of active ingredient and emulsified solvent in the water. E.g, propiconazole,
difenoconazole, etc.
Wettable powder (WP)
Wettable powder is a very common formulation for most of the fungicides, which is used
for spray mixtures. The modern wettable powders are water-dispersible which have the
quality to wet easily and disperse well in water. They are also called as Water-Dispersible
Powders (WDP). The active ingredient is incorporated, usually at the rate of 30-80% with
a finely ground inert dust (filler) such as kaolin, a wetting agent and a suspending
emulsifying agent. E.g., Indofil M – 45, Blitox 50 WP, etc.
A highly developed type of water-dispersible powder is called as colloidal
powder, which is so finely divided that the individual particles will never sediment out. A
typical colloidal powder contains 5-50% active ingredient with non-ionic wetting agents,
thickening agents and a hydrophilic diluents (carrier).
Dusts (D)
Dust formulations usually contain 1-10% active ingredient for direct application in dry
forms. They are manufactured in such a way that they are light enough to be carried by a
slight breeze for a considerable distance. The finely divided particles of active ingredient

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are carried on a carrier particle. The commonly used carriers (diluents) are attapulgite,
kaolin, talc, pyrophylite, diatomaceous earth, bentonite, calcium silicate, hydrated silica,
calcium carbonate, magnesium carbonate, gypsum, lime, etc. E.g., Blimix 4 % dust,
Micop D 06, etc.
Granules (Pellets)
Pellets are the formulations of the fungicide with inert material formed into particles
about the size of coarse sugar. The granules normally contain 3-10% of the active
ingredient. Due to their size, the granules do not drift but have limited application being
confined to soil and seed treatments. Granules have the advantage they can be measured
in dry form more easily and acurately than dusts or wettable powders. E.g., Topgun
50WG, Cosavit, Wokovit, Cumulas 80 WG, etc.
Suspension or slurries
A dry form of the active ingredient is mixed with a liquid in these formulations. Such
formulations usually contain a high percentage of active ingredients similar to Wettable
powders. They are mixed with water for final use and require agitation. These are mostly
used as seed dressers in seed processing companies. E.g., Flovin 35 SC, Cumal L 27 SL,
Share 40 SC.
Solutions
True solutions are formulations in which active ingredient or a combination of active
ingredients and a solvent is dissolved in water. Solutions have the advantage of requiring
no agitation after formulation is added in water. Now a day, the manufacturers are
concentrating to develop new formulations to increase the efficacy of the chemicals.
Some new formulations developed are soluble liquid (SL, e.g., Cumal L 27 SL and Casu
– B 3 SL), Soluble powder (SP), Water-soluble concentrate (WSC), Suspension
Concentrate (SC), e.g., Share 40 SC and Contaf plus 5 SC and Aqua Flow (AF).
Adjuvants
The fungicides can be commonly applied as either spraying or dusting. In spraying
method, the toxicant is made into a suspension in water. In order to increase the efficacy
of the water mixed sprays, certain substances like wetting agents, dispersing agents,
spreaders, stickers, etc. are added during the formulation of fungicides. These auxiliary
spray material are also called as adjuvants, which are usually inert materials added to
improve the physical characteristics of the toxicant and its carrier. Most of the material
used are surface-active agents and therefore induce variation in either surface tension or
interfacial tension. The various adjuvants are grouped as follows.
Dispersing agents (Deflocculating agents)

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These are the substances, which keep fine particles away from each other to prevent
deflocculation. These materials, when added to formulations, ensure uniform suspension
and retard sedimentation of particles in the spray suspension. These are also called as
deflocculating agents. E.g., Gelatin, plant gums and milk products.
Emulsifying agents
In powders (WDP), the active ingredient is incorporated, usually at the rate of 30-80%,
with a finely ground inert dust (filler) such as kaolin, a wetting agent and a suspending
agent. The commonly used suspending agents are sodium lignin sulphonate (Sulphite
dye), methylcelluloses, polyvinyl acetate and aluminum silicate. In addition, spreader-
sticker is sometimes desirable, especially on plants with glossy or waxy leaves. Agitation
is generally necessary to keep uniform suspension.
A typical colloidal powder contains 5-50% active ingredient with non-ionic wetting
agents (1-10% polyethylene oxide condensate), thickening agents like carboxy
methylcellulose and a hydrophilic diluents (carrier) such as bentonite.
Many surface-active substances like soap function as emulsifying agent, which retard the
settling out of droplets of water immiscible liquids like oils. This helps in uniform mixing
of substances in water suspensions.
Wetting agents (Wetters)
These are the material, which are added to ensure that there will be no layer of air
between a solid and a liquid as they reduce the surface tension of the particles. Wetting
agents, when added to aqueous fungicidal preparation, help in easy deposition on leaves.
E.g., Polyethylene oxide condensate, esters of fatty acids and flour.
Spreading agents (Spreaders)
Spreaders are the materials added to establish improved contact between the spray
materials and plant surface and thus ensuring a good coverage of fungicide. Wetting must
precede spreading and this is the only distinction between wetting and spreading.
Spreaders also reduce the surface tension and thus improve contact. E.g., soap, flour,
sulphate amines, soap amines, mineral oils, glyceride oil, terpene oil, resonates and
petroleum sulphonic acids.
Stickers (Adhesives)
The material, which are added to spray or dust to improve the adherence to plant surfaces
are called as stickers. They increase the tenacity of the fungicidal preparations, thus
increasing the residual action. E.g., Polyvinyl acetate, polybutanes, fish oil, linseed oil,
milk casein, gelatin, dextrines, polyethylene polysulphide, starch, gum arabic,

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hydrocarbon oils and bentonite clays. Milk casein and gelatin also act as good spreading
and wetting agents besides acting as stickers.
Safeners: A Chemical, which reduces the phytotoxicity of another chemical, is called
safener. E.g., copper sulphate is phytotoxic to plants, but with addition of lime its toxicity
is reduced. Lime is, therefore, a safener. Lime is used universally with chemicals to
prevent the formation of, or to neutralise arsenic, which is phytotoxic to plants. Glycerin
oils are also used as safeners.
Toxicity levels of chemicals: The toxicity levels of the fungicidal formulations are based
on the IC50 values. IC50 means the concentration of the chemical at which 50 per cent
growth of the test organisms is inhibited. The toxicity levels of the chemicals in all
formulations as coloured triangle.
IC50 Value
Triangle Colour Toxicity Level (mg/kg body weight)
Oral Dermal
Red Extremely toxic 1-50 1-200
Yellow Highly toxic 51-500 201-2000
Blue Moderately toxic 501-5000 2001-20000
Green Slightly toxic (Least > 5000 > 20000
toxic)
IC = Inhibitory concentration
Exercise:
1. Observe the types of fungicide formulations available in the market, test their
solubility, mix ability, sedimentation, etc. in the solution.
2. Observe different adjuvants being used for preparation of various formulations.

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EXERCISE NO. 14
METHODS OF APPLICATION OF FUNGICIDES I

(Foliar application)
Object: To know about different methods of application of fungicides on the foliage of

plants.
Proper selection of a fungicide and its application at the correct dose and at the
proper time are essential for the effective management of plant disease. In the application
method, the fungicide is delivered to the target (plant parts) where the active ingredient
acts.
The fungicidal application varies according to the nature of the host part diseased
and nature of survival and spread of the pathogen.
Foliar application
i. Spraying
This is the universal method to apply fungicides on the foliage. Spraying of
fungicides is done on leaves, stems, inflorescence and fruits. Spraying gives better
coverage on the plant surface. Loss due to drift and pollution risk is very less. It has long
residual effect. Spraying can be done at relatively high wind velocities. Spraying leads to
less hazards to the operators than dusting. Wettable powders and emulsifiable
concentrates are commonly used for preparing spray solutions. The most common
diluent or carrier is water. The dispersion of the spray is usually achieved by its passage
under pressure through nozzle of the sprayer. The amount of spray solution required for a
hectare of land depends on the nature of crops to be treated. For trees and shrubs, more
amount of spray solution is required. Depending on the volume of fluid used for
coverage, the sprays are categorized into high volume, medium volume, very low volume
and ultra low volume (ULV).
The spraying is done with the help of knapsack, power sprayer, HTP, etc.
Quantity of spray solution required (litre /ha)

Type of Spray
Ground crops Orchard crops / Trees
High volume 700 1200
Medium volume 400 to 700 800 to 1200
Low volume 225 to 400 350 to 800
Very low volume 60 to 225 250 to 350
Ultra low volume 20 50

lOMoARcPSD|10111315
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Preparation of spray solution

Usually Wettable powders or emulsifiable concentrates are diluted with water and
the resultant solution is used for spraying. A known quantity of the fungicidal formulation
is taken in a container to which a small quantity of water is added and mixed. Then the
required quantity of water is added to make the required concentration and stirred well. It
is taken in the sprayer and applied either in the morning or in the evening. For crops,
which have glossy leaf surface (banana) or hairy leaf surface (sugarcane, rice, etc)
spreading agents like soap (khadi soap at 1 g/litter of spray solution) or sticker (1 ml/litre) is
added in the spray solution. The required concentration of the fungicide should be prepared. If it
is less than the required concentration the targeted disease is not effectively controlled and if it
is more, the chemical may cause phytotoxicity to the crop. Generally the following
concentrations are used for the management of plant diseases.
Concentration Chemical required per
Fungicide
recommended one litre of water
Propiconazole 0.05% 0.50 ml/g
Carbendazim 0.1% 1.0g
Mancozeb 0.2% 2.0g
Copper oxychloride 0.25% 2.5g
Wettable sulphur 0.3% 3.0g
Requirement of Chemical
For spraying one acre of field crop generally 200 litres of spray solution (500
litres/ha) is required. The requirement of chemical for this area varies with the
concentration of the solution and number of sprayings recommended. E.g., for two
spraying in 2.0 ha of crop area with carbendazim (0.1%), the following is the calculations
for the fungicide requirement.
Carbendazin 1 g/litre of water 0.1%
Hence, 500 g/500 litre of water/ha 0.1%
For 2.0 ha. = 2 x 500 L of water = 1000 g/2ha for one spray.
For two sprays= 1000 g x 2 = 2000g
Hence, 2.0 kg of carbendazim is required for two sprays on two ha area.
ii. Dusting
Dusting requires less labour for the operation. Dusting covers more area in a day
than with spraying. Dusting equipments are lighter in weight and there is less risk of
corrosion. Dusts are applied to all aerial parts of a plant as an alternative to spraying. Dry
powders are used to cover the host surface. Generally, dusting is practiced when the plant
surface is wet with dew or raindrops. It is made during early hours in a day. Dust is
applied @ 25 kg/ha, e.g., sulphur dust to control powdery mildew diseases of different
crops in rainfed areas. The equipments employed for the dusting operation are bellows
duster, rotary duster, motorized knapsack duster and aircraft.
Exercise:
1. Prepare 0.2 and 0.3 per cent solutions of copper oxychloride for spraying.
2. Prepare 100 ppm streptomycin solution for spraying
*****************

lOMoARcPSD|10111315
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METHODS OF APPLICATION OF FUNGICIDES II

(Seed treatment and soil application)
Object: To know about different methods of seed treatment and soil application of
fungicides.
I) Seed treatment with chemicals

The seed treatment with fungicides is essential since the pathogens are carried on
or in the seed. Seed treatment protects the germinating seeds and growing seedlings
from soil-borne pathogens. Seed treatment is an effective and economic method of
disease control. Seed treatment is therapeutic when it kills pathogens that infect
embryos, cotyledons or endosperms under the seed coat, eradicative when it kills
pathogens that contaminate seed surfaces and protective when it prevents penetration
of soil-borne pathogens into the seedlings.
Using fungicides on seed is one of the most efficient and economical method.
Seed treatment chemicals may be seed disinfectant or seed protectant.
Seed disinfectant: Seed disinfectant, disinfects the seed but may not remain active
for a longer period after the seed has been sown.
Seed protectant: Seed protectant, disinfects the seed surface and stick to the seed
surfaces for sometimes after the seed has been sown, and thus giving temporary
protection to the young seedlings against soil-borne fungi. Systemic fungicides
disinfect the deep-seated infections in the seed.
The seed dressing chemicals may be applied by
a. Dry treatment
b. Wet treatment
c. Slurry
a. Dry seed treatment
In this method, the fungicide adheres in a fine form on the surface of the seeds. A
calculated quantity of fungicide is applied and mixed with seed using machinery
specially designed for the purpose. The fungicides may be treated with seeds of small
lots using simple rotary seed dresser (seed treating drum) or of large seed lots at seed
processing plants using grain-treating machines. Normally at field level, dry seed
treatment is carried out in rotary seed treating drums, which ensures proper coating of
the chemical on the surface of seeds. The little quantity of gum may be added for
better adherence of chemical on the seed surface. Dry seed dressing method is also
followed in pulses, cotton and oilseeds with the antagonistic fungi like Trichoderma
viride at the rate of 4g/kg of seed.
Dry seed treatment in rice: Mix a required quantity of fungicide with the required
quantity of seeds in a seed treating drum or polythene-lined gunny bag and mix

lOMoARcPSD|10111315
65
thoroughly to provide uniform coating of the fungicide over the seeds. Treat the seeds
24 hr prior to soaking for sprouting. Thiram, captan, carboxin, Tricyclazole, and
carbendazim are treated at 2g/kg of seed.
b. Wet seed treatment
This method involves preparing fungicide suspension in water and dipping the
seeds, seedlings, or propagative materials in it for a specified time.
Seed dipping in rice: Fungicidal solution is prepared by mixing any of the
recommended fungicides viz., carbendazim or pyroquilon or tricyclazole at 2g/litre of
water and seeds are soaked in the solution for 2 hrs. Drain the solution and keep the
seeds for sprouting.
Seed dipping in wheat: Prepare 0.2% solution of carboxin (2g/litre of water), soak
the seeds for six hrs, drain the solution and dry the seeds and use for sowing. It
eliminates the loose smut pathogen, Utsilago tritici.
Seed dip/root dip: The seedlings of fruits and vegetables are normally dipped in
0.25% copper oxychloride (2.5 g/litter of water) or 0.1% carbendazim solution
(1g/litre of water) for 5 to 10 minutes to protect against seedling blights and rots.
Rhizome dip: The rhizomes of cardamom, ginger and turmeric are treated with 0.1%
Emisan solution for 20 minutes to protect them from the attack by the pathogens
present in the soil.
Set dip/sucker dip: The sets of sugarcane and tapioca and suckers of pineapple ae
dipped in 0.1% Emisan solution or 0.1% carbendazim solution for 30 min.
c. Slurry treatment (Seed peleting): In this method, chemical is applied in the form of a
thin paste (active material is dissolved in small quantity of water) to the seed. The
required quantity of the fungicide slurry is mixed to the specified quantity of the seed,
so that during the process of treatment slurry get deposited on the surface of seeds in
the form of an thin paste, which later dries up. Seed processing units have usually
slurry treaters, which mix the fungicide slurry with specified quantity of seeds before
the seed lot is bagged.
Seed peleting in ragi: Mix 2.5g of carbendazim in 40 ml. of water and add 0.5g gum
to the fungicidal solution. Add 2kg of seeds to this solution and mix thoroughly to
ensure an uniform coating of the fungicide over the seed. Dry the seeds under shade
and sow after 24 hr after treatment.
d) Acid-delinting in cotton: This treatment helps to kill the seed-borne fungi and
bacteria in cotton. The seeds are treated with concentrated sulphuric acid @ 100
ml/kg of seed for 2-3 min. The seeds are then washed 2 or 3 times thoroughly with
cold water and shade dried. After drying, they are again treated with captan or thiram
@ 4g/kg of seed before sowing.

lOMoARcPSD|10111315
66
II) Soil application of chemicals

Chemical treatment of the soil is comparatively simple, especially when the soil is
fallow as the chemical is volatile and disappears quickly either by volatilization or
decomposition. Soil treating chemicals should be non-injurious to the plants in the
soil adjacent to the area where treatment has been carried out. Methods of soil
treatment are
a. Soil drenching b. Broadcasting c. Furrow application.
d. Fumigation and e. Chemigation.
a. Soil drenching: It is followed for controlling damping off and root rot infections at the
ground level. Required quantity of fungicide suspension is applied per unit area so
that the fungicide reaches to a depth of at least 10-15 cm. It is applied with water can
or through sprayer after removing nozzle.
b. Broadcasting: It is followed in granular fungicides wherein the pellets are broadcasted
near the plant.
c. Furrow application: It is done for the control of diseases where the direct application
of the fungicides on the plant surface leads to phytotoxicity. For the control of
powdery mildew of tobacco, sulphur dust is applied in the furrows and not on the
foliage.
d. Fumigation: Volatile (fumigants) toxicants such as methyl bromide, chloropicrin,
formaldehyde and vapam are the best chemical sterilants for soil to kill fungi and
nematodes. Soil fumigants penetrate the soil efficiently. Fumigations are normally
done in nursery areas and in glass house. The fumigant is applied to the soil and
covered by thin polythene sheets for 5 to 7 days and then removed. E.g.,
Formaldehyde is applied at 350 to 500 ml/m2 (i.e. 40 to 50 ml formalin (36 – 40%)
per litre of water and applied @ 8-10 L/m2). The treated soil is irrigated and used for
sowing/transplanting two weeks after treatment. Vapam is normally sprinkled on the
soil surface and covered. Volatile liquid fumigants are also injected to a depth of 15
cm using soil injector guns. The fumigants are not applied in the standing crops as
they are injurious to them.
e. Chemigation: the fungicides are directly mixed in the irrigation water. It is normally
practiced when sprinkler or drip irrigation system is followed in a field or garden.
Exercise:
1. Carry out the dry and wet seed treatments with captan or thiram @ 0.3 and 0.5 per
cent to gram seeds.
2. Prepare 4.0 per cent solution of formalin and carry out the soil treatment having
five square meter area of raised bed.
3. Apply fungicide to sugarcane sets.
**************

lOMoARcPSD|10111315
67
METHODS OF APPLICATION OF FUNGICIDES III

(Post harvest and special methods)
Object: To know about different methods of fungicide application for increasing the self-
life of fruits after harvesting.
I) Post harvest application

Fruits and vegetables are largely damaged after harvest mostly by fungi and
bacteria. The use of chemical method is found to be economical and effective.
Fumigation, dipping them in solutions and wrapping of the harvested products with
fungicide impregnated waxed papers are the important methods adopted in control of post
harvest diseases. Fumigation is followed in limited cases. Further fumigants with the
desirable qualities like quick volatility, highly effective against microorganisms, non-
phytotoxic and non-tainting in nature are very few. Commercial fumigant commonly used
for this purpose is sulphur. In addition, tetrachloroethylene, dichloromethane, ammonia,
nitrogen trichloride are also used as fumigants.
Dipping the fruits in fungicidal solution is better than in suspension. In suspension
the fungicide deposits on the surface of fruits. E.g., sodium –D-nphenylphenate solution
with 1% hexamine is suggested for oranges and apples. A dip in 0.1 - 0.2 per cent
aqueous solution of dichloran (2,6-dichloro 4 nitro aniline) for 1 or 2 min is effective
against Rhizopus rot of peaches.
Wrapping citrus fruits with waxed paper impregnated with diphenyl or
phenlbenzene or lining the packing container with the paper are followed in fruit industry
to avoid infection of Penicillium, Botrytis, Rhizopus spp. At present, post harvest
treatment of fruits and vegetables with systemic fungicides like benomyl or thiabendazole
is being practiced.
II) Special methods
i. Corm injection
Corm injection with the fungicide carbendazim is followed to control the dreadful
disease of banana viz., Panama wilt (Fusarium oxysporum f.sp. cubense).
Carbendazim is applied either in the form of 2 per cent solution or in the form of
capsule.
ii. Capsule application method: Carbendazim 50% WP 50 to 60 mg is taken in a
gelatinaceous capsule. The soil above corm in the banana tree is removed to expose
the corm. A capsule applicator made of iron is employed to make a hole to apply the
carbendazim capsule. Capsule applicator is made with 7 mm thickness iron rod. The
length of the rod is 45 cm. One end of the rod is sharpened. An iron plate is welded 7
cm above the pointed end for the purpose of inserting the rod at a constant depth of
the corm. At the top of the iron rod, a wooden handle is fixed. The iron rod is inserted
up to the iron plate into the exposed corm at an angle of 45º to make a hole.

lOMoARcPSD|10111315
68
Carbendazim capsule is pushed to the hole and covered with clay soil. The corm is
again covered with the removed soil.
iii. Application of carbendazim 2% solutions: Two per cent solution of carbendazim
50 WP is prepared by mixing 20 g of carbendazim in one litre of water. A whole in
the banana corm is made by following the same method as described above for
capsule application. Three ml carbendazim (2%) solution is injected into the hole
through syringe and the hole is plugged with clay soil.
iv. Root feeding.
Root feeding with the antibiotic, Aureofungin-sol + copper sulphate is employed in
the control of basal stem rot (Tanjore wilt or Ganoderma wilt) of coconut caused by
Ganoderma lucidum. Tridemorph (Calixin) can also be used at 2ml/100ml water. It is
an improved and effective method compared to trunk injection, which is an old and
dangerous method, which may cause even copper sulphate death of the tree.
Methodology: One pencil thickness, active coconut root is selected and exposed
outside from the soil by removing a layer of soil and is cut at a distance of 60 cm
from the tree. A slanting cut is made at the tip to have more surface area for
absorption. Root feeding chemical is prepared as follows:
Aureofungin-sol - 1.3 g
Copper sulphate - 0.5 g
Water - 100 ml
Copper sulphate is powdered well and it is dissolved in 100ml of water along with
the Aureofungin-sol. The solution is taken in a polythene bag. The tip of the root is
inserted into the polythene bag containing 100 ml of the fungicidal solution. Later the
mouth of the bag is tied tightly around the root keeping the cut end touching the
bottom portion of the bag. The solution is normally absorbed within 24 hrs. If it is not
absorbed in 24 hrs, another healthy root should be selected as done before and the
root feeding is given. It is followed thrice in a year at every four months for effective
control of the disease.
Exercise:
1. Prepare 0.05 and 0.10 per cent solutions of carbendazim, carry out fruit dip
treatment and record the self-life of the fruits.
2. Prepare the solution of Aureofungin or Calixin as indicated above and do the root
feeding exercise in coconut.
3. Prepare the capsules of carbendazim, apply to few healthy banana plants and
record disease observations once at capsule application and second at one month
after application.
*********************

lOMoARcPSD|10111315
69
EXERCISE NO. 15
CALCULATION OF FUNGICIDES: SPRAY CONCENTRATION
Object: To prepare proper concentration of fungicide spray solution.
Preparation of spray solution

Usually Wettable powders or emulsifiable concentrates are diluted with water and the resultant
solution is used for spraying. A known quantity of the fungicidal formulation is taken in a container to
which a small quantity of water is added and mixed. Then the required quantity of water is added to
make the required concentration and stirred well. It is taken in the sprayer and applied either in the
morning or in the evening. For crops, which have glossy leaf surface (banana) or hairy leaf surface
(sugarcane, rice, etc) spreading agents like soap (khadi soap at 1 g/litter of spray solution) or sticker (1
ml/litre) is added in the spray solution. The required concentration of the fungicide should be
prepared. If it is less than the required concentration the targeted disease is not effectively controlled
and if it is more, the chemical may cause phytotoxicity to the crop. Generally the following
concentrations are used for the management of plant diseases.
Concentration Chemical required per
Fungicide
recommended one litre of water
Propiconazole 0.05% 0.50 ml/g
Carbendazim 0.1% 1.0g
Mancozeb 0.2% 2.0g
Copper oxychloride 0.25% 2.5g
Wettable sulphur 0.3% 3.0g
Requirement of Chemical
For spraying one acre of field crop generally 200 litres of spray solution (500
litres/ha) is required. The requirement of chemical for this area varies with the
concentration of the solution and number of sprayings recommended. E.g., for two
spraying in 2.0 ha of crop area with carbendazim (0.1%), the following is the calculations
for the fungicide requirement.
Carbendazin 1 g/litre of water: 0.1%

Hence, 500 g/500 litre of water/ha 0.1%
For 2.0 ha. = 2 x 500 L of water = 1000 g/2ha for one spray.
For two sprays = 1000 g x 2 = 2000g
Hence, 2.0 kg of carbendazim is required for two sprays on two ha area.
Exercise:
3. Prepare 0.2 and 0.3 per cent solutions of copper oxychloride for spraying.
4. Prepare 100 ppm streptomycin solution for spraying
*************

lOMoARcPSD|10111315
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EXERCISE NO. 16
COLLECTION AND PRESERVATION OF DISEASE SPECIMENS
Objectives:
1. To get acquainted with the different methods of preservation and maintenance of
disease samples and cultures.
2. To know the importance of disease specimen and culture collection.
3. To better understand diseases with special reference to the symptoms produced by
the pathogens and
4. To conduct systematic mycological work.
1. HERBARIUM COLLECTION, PRESERVATION AND MAINTENANCE OF

SPECIMENS
Preservation of diseased materials (herbariums) on their natural substrates as dry
specimens or wet specimens is essential for conducting systematic mycological work and
important taxonomic' research on various microorganisms. The collection of these
diseased specimens in various repositories not only help to maintain their identity but are
also essential for various pathological aspects such as genetic variability, effect of
biodiversity and on various mycological aspects of different isolates of a pathogens.
Preservation means killing or stopping the growth of an organism in or on the substrate
on which it grows.
(i) Dry preservation of specimens
The following steps are followed for dry specimen preservation.
a. Collection
The representative sample collected should be of good quality preferably, should
have reproductive parts, and be in sufficient quantity.
1. Disease material should be collected in the field and then it is properly pressed
between the blotter sheets.
2. Paper slips or bags are used to indicate locality, host, date and place of collection.
Mention should be made if there is any other substrate on which it is present.
3. Collections of damp and fleshy material should be properly wrapped in moisture
absorbing sheets to avoid their damages by moulds, insects, etc.
4. Dry specimens (e.g. flowering plant parasites) may be kept for a very long period.
b. Drying and accession
In drying process, dehydration of collected material is done. This helps in long-
term preservation. The process is carried out in hot air or in the sunlight for a few days
after carefully pressing the specimens between blotter sheets. Frequent changes of blotter
sheets help proper dehydration of the material.

lOMoARcPSD|10111315
71
c. Labeling and packing

The accessioned material is kept in good quality herbarium packet (6 X 4"). This
is attached to a chart paper sheets (16.5 X 10.5"). On the extreme lower left of the chart
paper, entries regarding the name of the pathogen, host (if applicable), locality, date and
name of the scientist who identifies the specimen are mentioned on a printed level (5 x
3") which is already pasted there.
d. Disinfection and storage
These folders along with the specimens are fumigated with methyl bromide
vapours in a fumigation chamber for 24-48 hours before storage in wooden or steel
cupboards to protect them from moulds, insect, mites or from climatic or environmental
hazards. Naphthalene balls, xylene, thymol or flit may be sprayed in compartments of
cupboards whenever the methyl bromide fumigation is not available. This helps the
preservation of the specimens without affecting their originality for years. Fumigation
process may be carried out periodically to avoid damages by mites and insects.
(ii) Wet preservation of specimen
First the wet sample is washed off under running water, then specimen is placed
in the 5 % formalin solution. The formalin solution is frequently changed
(iii) Colour preservation
This is accomplished by boiling plant parts in a mixture of one part of glacial
acetic acid and four parts of water till the green colour reappears and then keeping it in
5% copper sulphate solution for at least six and not more than twenty-four hours. This
material is then washed for several hours in running tap water and preserved in 1.5%
aqueous solution of sulphurous acid or treat the material 6-24 hours in 5% solution of
copper sulphate and wash in running tap water for 2 hours. Immerse in 5-6% solution of
sulphuric acid for 15 days and then mount on a plaque. Cover with convex glass, seal
with a mixture of 300g tar, 60g virgin wax, and one part of anhydrous lanolin. Coloured
fruits are preserved in Hesler's preservative containing 50 g ZnCl 2, 25 ml formaldehyde
and 25 ml glycerin in one liter of distilled water.
2. STORAGE OF FUNGAL SPECIMENS
It has been found that during storage of dried diseased specimens as well as
culture types, one faces several climatic and other environmental hazards, e.g. humidity,
mites, moulds and insects which spoil the specimens off and on. In such cases volatile
substances like xylene, naphthalene, paradichlorobenzene, tractor oil, thymol and flit can
easily be used for repelling mites and insects. These are quite easy to handle and do not
spoil the specimens.
3. IDENTIFICATION SERVICE
The basic and most important requisites for identification are : (i) the proper
specimens, (ii) accurate description, and (iii) illustration derived from them. The
identification of the specimens is done by matching of good specimens that have already
been correctly named.
************

Path-121-Practical C

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lOMoARcPSD|10111315

Plant Pathology (Mahatma Phule Krishi Vidyapeeth)

StuDocu is not sponsored or endorsed by any college or university

2. The laboratory instruments such as microscope, micrometers, electrically

1. All figures must be drawn with drawing pencil.

3. Assessment of the practical will be done on the basis of attendance in the

4. If a student remains absent for a practical, the assessment of exercise will be

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ACQUAITANCE WITH VARIOUS LABORATORY EQUIPMENTS AND

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 Time is counted once the desired temperature is achieved in the oven.

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The cabinet is fabricated out of thick board of sunmica or is of stainless steel.

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• Open stream cock and light gas or switch on electricity.

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There are two types of microscope in common use.

e.g. Eye piece (10x) objective (44x) = 440.

Mounting of cultures and specimens for microscopic investigation

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Instruments used in Laboratory of Plant Pathology and their uses

Sr. Name of Instrument Use

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I. STUDY OF TYPICAL FUNGUS :

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Sporangiole : A small sporangium without columella.

II. VEGETATIVE STRUCTURES :

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Vascular mycelium : When the mycelium is confined to or found to grow in vascular

III. MODIFICATIONS OF MYCELIUM

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REPRODUCTIVE STRUCTURES AND SPORE FRUITS IN FUNGI

Asexual Sexual Vegetative

Endogenous Exogenous Zygote Chlamydospores

ASEXUAL SPORES IN FUNGI

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These spores are borne externally on sporophores.

e.g. conidia, oidia, etc.

Conidia (Sing. Conidium) :

The conidia are produced asexually on a specialized hyphae or stalk known as

Oidia (Sing. Oidium)

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1. Coremium or Synnemata (Pl. Coremia):

2. Sporodochium (Pl. Sporodochia):

3. Pycnidium (Pl. Pycnidia):

4. Acervulus (Pl. acervuli):

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It is compact mass of hyphae giving rise to short, simple, hyaline

5. Sorus (Pl. sori):

6. Aecium (Pl. aecia) :

7. Pycnium or Spermagonium (Pl. Pycnia) :

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SEXUAL SPORES IN FUNGI

It is the result of union between female and male

Zygospores are formed by the union of two

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phylum Zygomycota, class-Zygomycetes and Trichomycetes, order Mucorales and

Ascospores are produced in a sac known as ascus which

These are the sexual spores produced on club shaped